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Abstract:

The inventors have made the surprising discovery that SGK1, a
serine/threonine kinase previously described as being involved in
regulation of cellular sodium homeostasis, has a novel and unexpected
function in the differentiation and function of a specific subset of CD4
T cells, the TH17 lineage. Described herein are methods and compositions
for modulation of TH17 cell differentiation, proliferation, and/or
function that rely upon modulating the activity or expression of SGK1.
Such methods and compositions are useful in the treatment of disorders
including autoimmune diseases, chronic inflammatory conditions,
infectious diseases, and cancer.

Claims:

1. A method of inhibiting differentiation of a CD4.sup.+ T cell or a
CD4.sup.+ T cell population into a TH17 cell or TH17 cell population, the
method comprising contacting a CD4.sup.+ T cell or CD4.sup.+ T cell
population with a serum and glucocorticoid-regulated kinase 1 (SGK1)
inhibitor in an amount sufficient to inhibit TH17 cell differentiation.

2. The method of claim 1, further comprising contacting the CD4.sup.+ T
cell or CD4.sup.+ T cell population with an inhibitor or antagonist of
one or more of the following molecules: TGF-.beta., IL-6, IL-21, IL-23,
RORγt, RORα, STAT3, IRF4, AhR (aryl hydrocarbon receptor),
and BATf.

3. A method of inhibiting a TH17 cell-mediated immune response in a
subject in need thereof, the method comprising administering to a subject
in need thereof a therapeutically effective amount of a serum and
glucocorticoid-regulated kinase 1 (SGK1) inhibitor to inhibit a TH17
cell-mediated immune response.

4. The method of claim 3, wherein the TH17 cell-mediated response being
inhibited comprises expression or production of IL-17 by a TH17 cell.

5. The method of claim 3, wherein the TH17 cell-mediated response being
inhibited comprises expression or production of one or more of IL-17F,
IL-22, IL-26, IL-21, and TNF-.alpha..

6. The method of claim 3, wherein the TH17 cell-mediated response being
inhibited comprises inhibition of proliferation of or expansion of a TH17
cell.

7. The method of claim 3, wherein the TH17 cell-mediated response being
inhibited comprises trafficking of a TH17 cell.

8. The method of claim 3, wherein the subject in need of inhibition of a
TH17-mediated immune response has a TH17-mediated disorder.

9. The method of claim 8, wherein the TH17-mediated disorder is an
autoimmune disease or a chronic inflammatory disease.

11. The method of claim 3, wherein the SGK1 inhibitor is a small
molecule, a blocking antibody or antigen-binding fragment thereof, a
polypeptide, an antisense oligonucleotide, an RNA molecule, an aptamer,
or a ribozyme.

12. The method of claim 11, wherein the small molecule is a small
molecule of Formula (I): ##STR00055## wherein R1 is optionally
substituted phenyl, optionally substituted β-napthyl, or optionally
substituted 3-CN-phenyl; wherein R2 is CO2R4 or C(R4,R5) CO2R4;
wherein R3 and R4 are independently absent, H, C1-C6 alkyl, or
C5-C8 cycloalkyl; each of which may be optionally substituted;
wherein R5 and R6 are independently absent, H, or C1-C6 alkyl,
each of which may be optionally substituted; and pharmaceutically
acceptable salts thereof.

13. The method of claim 11, wherein the small molecule is a small
molecule of Formula (Ia): ##STR00056## Formula (Ia)

14. The method of claim 13, wherein R1 is phenyl, R2 is CO2H, and R3
is H.

15. The method of claim 13, wherein R1 is phenyl, R2 is CO2H, and R3
is ##STR00057##

16. The method of claim 13, wherein R1 is phenyl, R2 is CO2H, and R3
is ##STR00058##

17. The method of claim 13, wherein R1 is phenyl, R2 is CO2H, and R3
is ##STR00059##

18. The method of claim 13, wherein R1 is β-napthyl, R2 is
CH2CO2H, and R3 is H.

19. The method of claim 13, wherein R1 is β-napthyl, R2 is
##STR00060## and R3 is H.

20. The method of claim 13, wherein R1 is β-napthyl, R2 is
##STR00061## and R3 is H.

21. The method of claim 13, wherein R1 is phenyl, R2 is ##STR00062##
and R3 is H.

22. The method of claim 13, wherein R1 is 3-CN-phenyl, R2 is
##STR00063## and R3 is H.

24. The method of claim 3, further comprising administering to the
subject in need thereof a therapeutic agent selected from the group
consisting of a cytokine inhibitor, a growth factor inhibitor, a
chemotherapeutic agent, an immunosuppressant, an anti-inflammatory agent,
a metabolic inhibitor, an enzyme inhibitor, a cytotoxic agent, and a
cytostatic agent.

25.-45. (canceled)

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. §119(e) of
U.S. Provisional Application No. 61/230,376 filed Jul. 31, 2009, the
contents of which are herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The invention relates to compositions and methods for modulating
TH17 responses.

BACKGROUND

[0004] TH17 cells are a subset of CD4+ T cells that are abundant at
mucosal interfaces, where they can contain infection with pathogenic
bacteria and fungi (Weaver et al., 2007). TH17 cells produce IL-17A (also
referred to as IL-17), IL-17F, and IL-22, cytokines involved in
neutrophilia, tissue remodeling and repair, and production of
antimicrobial proteins. TH17 cells differentiate in response to the
STAT3-activating cytokines IL-6, IL-21, and IL-23, along with TGF-β
and IL-1β (Korn et al., 2009).

[0006] The inventors have made the surprising discovery that SGK1, a
serine/threonine kinase previously described as being involved in
regulation of cellular sodium homeostasis, has a novel and unexpected
function in the differentiation and function of a specific subset of CD4
T cells, the TH17 lineage. Described herein are methods and compositions
for modulation of TH17 cell differentiation, proliferation, and/or
function that rely upon modulating the activity or expression of SGK1.

[0007] Accordingly, in one aspect described herein are methods of
inhibiting differentiation of a CD4+ T cell or a CD4+ T cell
population into a TH17 cell or TH17 cell population. Such methods
comprise contacting a CD4+ T cell or CD4+ T cell population
with a serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitor in an
amount sufficient to inhibit TH17 cell differentiation. In some
embodiments, the methods of inhibiting differentiation into a TH17 cell
or TH17 cell population further comprise contacting the CD4+ T cell
or CD4+ T cell population with an inhibitor or antagonist of one or
more of the following molecules: TGF-β, IL-6, IL-21, IL-23,
RORγt, RORα, STAT3, IRF4, AhR (aryl hydrocarbon receptor),
and BATf.

[0008] In another aspect, described herein are methods of inhibiting a
TH17 cell-mediated immune response in a subject in need thereof. Such
methods comprise administering to a subject in need thereof a
therapeutically effective amount of a serum and glucocorticoid-regulated
kinase 1 (SGK1) inhibitor to inhibit a TH17 cell-mediated immune
response. In some embodiments of these methods, the TH17 cell-mediated
response being inhibited comprises expression or production of IL-17 by a
TH17 cell. In some embodiments of these methods, the TH17 cell-mediated
response being inhibited further comprises expression or production of
one or more of IL-17F, IL-22, IL-26, IL-21, and TNF-α.

[0009] In some embodiments of these methods, the TH17 cell-mediated
response being inhibited comprises inhibition of proliferation of or
expansion of a TH17 cell. In some embodiments of these methods, the TH17
cell-mediated response being inhibited comprises trafficking of a TH17
cell.

[0011] In some embodiments of these methods, the SGK1 inhibitor is a small
molecule, a blocking antibody or antigen-binding fragment thereof, a
polypeptide, an antisense oligonucleotide, an RNA molecule, an aptamer,
or a ribozyme.

[0012] In some embodiments, the small molecule is a small molecule of
Formula (I):

##STR00001##

wherein R1 is optionally substituted phenyl, optionally substituted
β-napthyl, or optionally substituted 3-CN-phenyl; wherein R2 is
CO2R4 or C(R4,R5) CO2R4; wherein R3 and R4 are independently
absent, H, C1-C6 alkyl, or C5-C8 cycloalkyl; each of
which may be optionally substituted; wherein R5 and R6 are independently
absent, H, or C1-C6 alkyl, each of which may be optionally
substituted; and pharmaceutically acceptable salts thereof.

[0013] In some embodiments, the small molecule is a small molecule of
Formula (Ia):

##STR00002##

[0014] In some embodiments, R1 is phenyl, R2 is CO2H, and R3 is H. In
some embodiments, R1 is phenyl, R2 is CO2H, and R3 is

##STR00003##

In some embodiments, R1 is phenyl, R2 is CO2H, and R3 is

##STR00004##

[0015] In some embodiments, R1 is phenyl, R2 is CO2H, and R3 is

##STR00005##

In some embodiments, R1 is β-napthyl, R2 is CH2CO2H, and
R3 is H. In some embodiments, R1 is β-napthyl, R2 is

[0017] In some embodiments of these methods, the method of inhibiting a
TH17-mediated immune response further comprising administering to the
subject in need thereof a therapeutic agent selected from the group
consisting of a cytokine inhibitor, a growth factor inhibitor, a
chemotherapeutic agent, an immunosuppressant, an anti-inflammatory agent,
a metabolic inhibitor, an enzyme inhibitor, a cytotoxic agent, and a
cytostatic agent.

[0018] In one aspect, described herein are uses of an SGK1 inhibitor in
inhibiting a TH17 cell-mediated immune response in a subject in need
thereof.

[0019] In some embodiments of the uses of SGK1 inhibitors, the TH17
cell-mediated response being inhibited comprises expression or production
of IL-17 by a TH17 cell. In some embodiments, the TH17 cell-mediated
response being inhibited further comprises expression or production of
one or more of IL-17F, IL-22, IL-26, IL-21, and TNF-α.

[0020] In some embodiments of the uses of SGK1 inhibitors, the TH17
cell-mediated response being inhibited comprises inhibition of
proliferation of or expansion of a TH17 cell. In some embodiments of the
uses of SGK1 inhibitors, the TH17 cell-mediated response being inhibited
comprises trafficking of a TH17 cell.

[0022] In some embodiments of the uses of SGK1 inhibitors, the SGK1
inhibitor is a small molecule, a blocking antibody or antigen-binding
fragment thereof, a polypeptide, an antisense oligonucleotide, an RNA
molecule, an aptamer, or a ribozyme.

[0023] In some such embodiments, the small molecule is a small molecule of
Formula (I):

##STR00010##

wherein R1 is optionally substituted phenyl, optionally substituted
β-napthyl, or optionally substituted 3-CN-phenyl; wherein R2 is
CO2R4 or C(R4,R5) CO2R4; wherein R3 and R4 are independently
absent, H, C1-C6 alkyl, or C5-C8 cycloalkyl; each of
which may be optionally substituted; wherein R5 and R6 are independently
absent, H, or C1-C6 alkyl, each of which may be optionally
substituted; and pharmaceutically acceptable salts thereof.

[0024] In some embodiments, the small molecule is a small molecule of
Formula (Ia):

##STR00011##

[0025] In some embodiments of the small molecule of Formula (Ia), R1 is
phenyl, R2 is CO2H, and R3 is H. In some embodiments, R1 is phenyl,
R2 is CO2, and R3 is

##STR00012##

In some embodiments, R1 is phenyl, R2 is CO2H, and R3 is

##STR00013##

In some embodiments, R1 is phenyl, R2 is CO2H, and R3 is

##STR00014##

In some embodiments, R1 is β-napthyl, R2 is CH2CO2H, and
R3 is H. In some embodiments, R1 is β-napthyl, R2 is

[0027] In another aspect, described herein are methods of modulating a
TH17 cell-mediated immune response in a subject in need thereof, such
methods comprising administering to a subject in need thereof a
therapeutically effective amount of an agent that modulates serum and
glucocorticoid-regulated kinase 1 (SGK1) activity to modulate a TH17
cell-mediated immune response.

[0028] In another aspect, described herein are methods of modulating TH17
cell-mediated cytokine production in a subject in need thereof, such
methods comprising administering to a subject in need thereof a
therapeutically effective amount of an agent that modulates serum and
glucocorticoid-regulated kinase 1 (SGK1) activity to modulate TH17
cell-mediated cytokine production.

[0029] In another aspect, described herein are methods of modulating TH17
cell proliferation in a subject in need thereof, such methods comprising
administering to a subject in need thereof a therapeutically effective
amount of an agent that modulates serum and glucocorticoid-regulated
kinase 1 (SGK1) activity to modulate TH17 cell proliferation.

[0030] In another aspect, described herein are methods of modulating TH17
cell-mediated inflammatory activity in a subject in need thereof, such
methods comprising administering to a subject in need thereof a
therapeutically effective amount of an agent that modulates serum and
glucocorticoid-regulated kinase 1 (SGK1) activity to modulate TH17
cell-mediated inflammatory activity.

[0031] In another aspect, described herein are methods of modulating TH17
cell migration in a subject in need thereof, such methods comprising
administering to a subject in need thereof a therapeutically effective
amount of an agent that modulates serum and glucocorticoid-regulated
kinase 1 (SGK1) activity to modulate TH17 cell migration.

[0032] In some embodiments of any of these methods, the method further
comprises administering to the subject in need another therapeutic agent
selected from the group consisting of a cytokine inhibitor, a growth
factor inhibitor, an immunosuppressant, an anti-inflammatory agent, a
metabolic inhibitor, an enzyme inhibitor, a cytotoxic agent, and a
cytostatic agent.

[0033] In some embodiments of these methods, the agent is an inhibitor of
SGK1 that decreases SGK1 activity. In some such embodiments of these
methods, the inhibitor of SGK1 is a an antibody or antigen-binding
fragment thereof, a polypeptide, a small molecule, an antisense
oligonucleotide, an RNA molecule, an aptamer, or a ribozyme.

[0034] In some embodiments, the small molecule is a small molecule of
Formula (I):

[0038] In other embodiments of these methods, the agent is an agonist of
SGK1 that increases SGK1 activity. In such embodiments, the agonist of
SGK1 activity is a an antibody or antigen-binding fragment thereof, a
polypeptide, a small molecule, or an activating RNA molecule.

[0044] In some embodiments of these methods of modulating SGK1 activity,
the subject has or is at risk of having an atopic condition. In some such
embodiments, the atopic condition is asthma, allergic rhinitis,
gastrointestinal allergy, atopic dermatitis, eosinophilia,
conjunctivitis, or eczema.

[0045] In one aspect, methods of modulating the differentiation of human
TH17 cells from a population of human naive CD4+ T cells are provided,
the methods comprising contacting a human naive CD4+ T cell with an
effective amount of an agent that modulates serum and
glucocorticoid-regulated kinase 1 (SGK1) activity.

[0046] In another aspect, methods of modulating the level of expression of
IL-17 from human naive CD4+ T cells are provided, such method comprising
contacting a human naive CD4+ T cell with an effective amount of an agent
that modulates serum and glucocorticoid-regulated kinase 1 (SGK1)
activity.

[0047] In another aspect, methods for modulating TH17 cell activity are
provided, the methods comprising contacting a human naive CD4+ T cell
with an effective amount of an agent that modulates serum and
glucocorticoid-regulated kinase 1 (SGK1) activity.

[0048] In another aspect, methods for modulating TH17 cell number are
provided, the methods comprising contacting a human naive CD4+ T cell
with an effective amount of an agent that modulates serum and
glucocorticoid-regulated kinase 1 (SGK1) activity.

[0049] In one aspect, methods of detecting TH17 cells in a test biological
sample are provided. Such methods comprise contacting a test biological
sample with a probe that detects a level of serum and
glucocorticoid-regulated kinase 1 (SGK1) relative to a control biological
sample, such that an increase in said level of SGK1 in the test
biological sample relative to the control biological sample is indicative
of the presence of TH17 cells in the test biological sample. In some
embodiments of these methods, the biological sample is selected from the
group consisting of blood sample, serum sample, cell sample, tissue
sample, bone marrow and biopsy.

DEFINITIONS

[0050] For convenience, certain terms employed herein, in the
specification, examples and appended claims are collected here. Unless
stated otherwise, or implicit from context, the following terms and
phrases include the meanings provided below. Unless explicitly stated
otherwise, or apparent from context, the terms and phrases below do not
exclude the meaning that the term or phrase has acquired in the art to
which it pertains. The definitions are provided to aid in describing
particular embodiments, and are not intended to limit the claimed
invention, because the scope of the invention is limited only by the
claims. Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs.

[0051] The term "agent" as used herein means any compound or substance
such as, but not limited to, a small molecule, nucleic acid, polypeptide,
peptide, drug, ion, etc. An "agent" can be any chemical, entity, or
moiety, including without limitation synthetic and naturally-occurring
proteinaceous and non-proteinaceous entities. In some embodiments, an
agent is a nucleic acid, a nucleic acid analogue, protein, antibody,
peptide, aptame, oligomer of nucleic acids, amino acid, or carbohydrate,
and includes, without limitation, proteins, oligonucleotides, ribozymes,
DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and
modifications and combinations thereof etc. In certain embodiments,
agents are small molecules having a chemical moiety. For example,
chemical moieties include unsubstituted or substituted alkyl, aromatic,
or heterocyclyl moieties. Compounds can be known to have a desired
activity and/or property, or can be selected from a library of diverse
compounds.

[0052] As used herein, the term "small molecule" refers to a chemical
agent which can include, but is not limited to, a peptide, a
peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a
polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an
organic or inorganic compound (e.g., including heterorganic and
organometallic compounds) having a molecular weight less than about
10,000 grams per mole, organic or inorganic compounds having a molecular
weight less than about 5,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 1,000 grams per mole,
organic or inorganic compounds having a molecular weight less than about
500 grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds.

[0053] The term "modulate" is used consistently with its use in the art,
e.g., meaning to cause or facilitate a qualitative or quantitative
change, alteration, or modification in one or more biological processes,
mechanisms, effects, responses, functions, activities, pathways, or other
phenomena of interest. Without limitation, such change may be an
increase, decrease, or change in relative strength or activity of
different components or branches of the process, mechanism, effect,
response, function, activity, pathway, or phenomenon. Accordingly, as
used herein "modulating" refers to a change of at least 10%, at least
about 20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least about
90%, up to and including a 100% change, or any change of at least about
2-fold, at least about 3-fold, at least about 4-fold, at least about
5-fold, at least about 10-fold, at least about 100-fold, at least about
1000-fold, or any modulation between 2-fold and 1000-fold, or greater, as
compared to a reference level. A "modulator" is an agent, such as a small
molecule or other agents described herein, that causes or facilitates a
qualitative or quantitative change, alteration, or modification in a
process, mechanism, effect, response, function, activity, pathway, or
phenomenon of interest.

[0054] The terms "decrease", "reduced", "reduction", "decrease" or
"inhibit" are all used herein generally to mean a decrease by a
statistically significant amount. However, for avoidance of doubt,
"reduced", "reduction" or "decrease" or "inhibit" means a decrease by at
least 10% as compared to a reference level, for example a decrease by at
least about 20%, or at least about 30%, or at least about 40%, or at
least about 50%, or at least about 60%, or at least about 70%, or at
least about 80%, or at least about 90% or up to and including a 100%
decrease (e.g. absent level or non-detectable level as compared to a
reference sample), or any decrease between 10-100% as compared to a
reference level.

[0055] The terms "increased", "increase" or "enhance" or "activate" are
all used herein to generally mean an increase by a statically significant
amount; for the avoidance of any doubt, the terms "increased", "increase"
or "enhance" or "activate" means an increase of at least 10% as compared
to a reference level, for example an increase of at least about 20%, or
at least about 30%, or at least about 40%, or at least about 50%, or at
least about 60%, or at least about 70%, or at least about 80%, or at
least about 90% or up to and including a 100% increase or any increase
between 10-100% as compared to a reference level, or at least about a
2-fold, or at least about a 3-fold, or at least about a 4-fold, or at
least about a 5-fold or at least about a 10-fold increase, or any
increase between 2-fold and 10-fold or greater as compared to a reference
level.

[0056] The term "statistically significant" or "significantly" refers to
statistical significance and generally means a two standard deviation
(2SD) difference relative to a reference. The term refers to statistical
evidence that there is a difference. It is defined as the probability of
making a decision to reject the null hypothesis when the null hypothesis
is actually true. The decision is often made using the p-value.

[0057] As used herein, the term "DNA" is defined as deoxyribonucleic acid.

[0058] The term "polynucleotide" is used herein interchangeably with
"nucleic acid" to indicate a polymer of nucleosides. Typically a
polynucleotide of this invention is composed of nucleosides that are
naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine,
cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and
deoxycytidine) joined by phosphodiester bonds. However the term
encompasses molecules comprising nucleosides or nucleoside analogs
containing chemically or biologically modified bases, modified backbones,
etc., whether or not found in naturally occurring nucleic acids, and such
molecules may be preferred for certain applications. Where this
application refers to a polynucleotide it is understood that both DNA,
RNA, and in each case both single- and double-stranded forms (and
complements of each single-stranded molecule) are provided.
"Polynucleotide sequence" as used herein can refer to the polynucleotide
material itself and/or to the sequence information (e.g. the succession
of letters used as abbreviations for bases) that biochemically
characterizes a specific nucleic acid. A polynucleotide sequence
presented herein is presented in a 5' to 3' direction unless otherwise
indicated.

[0059] The term "polypeptide" as used herein refers to a polymer of amino
acids. The terms "protein" and "polypeptide" are used interchangeably
herein. A peptide is a relatively short polypeptide, typically between
about 2 and 60 amino acids in length. Polypeptides used herein typically
contain amino acids such as the 20 L-amino acids that are most commonly
found in proteins. However, other amino acids and/or amino acid analogs
known in the art can be used. One or more of the amino acids in a
polypeptide may be modified, for example, by the addition of a chemical
entity such as a carbohydrate group, a phosphate group, a fatty acid
group, a linker for conjugation, functionalization, etc. A polypeptide
that has a nonpolypeptide moiety covalently or noncovalently associated
therewith is still considered a "polypeptide". Exemplary modifications
include glycosylation and palmitoylation. Polypeptides may be purified
from natural sources, produced using recombinant DNA technology,
synthesized through chemical means such as conventional solid phase
peptide synthesis, etc. The term "polypeptide sequence" or "amino acid
sequence" as used herein can refer to the polypeptide material itself
and/or to the sequence information (e.g., the succession of letters or
three letter codes used as abbreviations for amino acid names) that
biochemically characterizes a polypeptide. A polypeptide sequence
presented herein is presented in an N-terminal to C-terminal direction
unless otherwise indicated.

[0060] The term "expression" refers to the cellular processes involved in
producing RNA and proteins and as appropriate, secreting proteins,
including where applicable, but not limited to, for example,
transcription, translation, folding, modification and processing.
"Expression products" include RNA transcribed from a gene, and
polypeptides obtained by translation of mRNA transcribed from a gene.
Expressing a cytokine, for example, can refer to RNA transcription of a
cytokine, translation of a cytokine, secretion of a cytokine, processing
of a cytokine, or any combination therein. Detecting expression of an RNA
transcript or polypeptide can be performed using any method known to one
of skill in the art, including, but not limited to, semi-quantitative and
quantitative RT-PCR, Northern blot analysis, Western blot analysis,
ELISA, bead arrays, chip arrays, and flow cytometry (including
intracellular detection of proteins, such as cytokine, by flow
cytometry).

[0061] As used herein, the term "target" refers to a biological molecule
(e.g., SGK1 peptide, SGK1 polypeptide, protein, lipid, carbohydrate) to
which an modulating agent, such as an inhibitor or an activator, can
selectively bind. The target can be, for example, an intracellular target
(e.g., an intracellular protein target) or a cell surface target (e.g., a
membrane protein, a receptor protein).

[0062] As used herein, "selectively binds" or "specifically binds" refers
to the ability of an activator or inhibitor, described herein, to bind to
a target, such as the SGK1 polypeptide, with a KD 10-5 M (10000
nM) or less, e.g., 10-6 M or less, 10-7 M or less, 10-8 M
or less, 10-9M or less, 10-10 M or less, 10-11 M or less,
or 10-12 M or less. For example, if an activator or inhibitor
described herein binds to the SGK1 polypeptide with a KD of
10-5 M or lower, but not to other molecules, or a related homologue,
then the agent is said to specifically bind the SGK1 polypeptide.
Specific binding can be influenced by, for example, the affinity and
avidity of the activator or inhibitor and the concentration of the
activator or inhibitor used. The person of ordinary skill in the art can
determine appropriate conditions under which the activators or inhibitors
described herein selectively bind using any suitable methods, such as
titration of an activator or inhibitor in a suitable cell binding assay.

[0063] The term "specificity" refers to the number of different types of
antigens or antigenic determinants to which a particular SGK1 modulating
agent can bind. The specificity of an SGK1 modulating agent can be
determined based on affinity and/or avidity. The affinity, represented by
the equilibrium constant for the dissociation (KD) of a target with
an SGK1 modulating agent (such as a small molecule or antibody or
antigen-binding fragment described herein), is a measure for the binding
strength between the target and the SGK1 modulating agent: the lesser the
value of the KD, the stronger the binding strength between an
antigenic determinant and the antigen-binding molecule. Alternatively,
the affinity can also be expressed as the affinity constant (KA),
which is 1/KD). As will be clear to the skilled person, affinity can
be determined in a manner known per se, depending on the specific target
of interest. Accordingly, an SGK1 modulating agent as defined herein is
said to be "specific for" SGK1 compared to a second target when it binds
to SGK1 with an affinity (as described above, and suitably expressed, for
example as a KD value) that is at least 10 times, such as at least
100 times, and preferably at least 1000 times, and up to 10.000 times or
more better than the affinity with which the modulating agent binds to
another target, such as SGK2.

[0064] As used herein, "immunoglobulin" refers to a family of polypeptides
which retain the immunoglobulin fold characteristic of antibody
molecules, which comprise two β sheets and, usually, a conserved
disulphide bond. Members of the immunoglobulin superfamily are involved
in many aspects of cellular and non-cellular interactions in vivo,
including widespread roles in the immune system (for example, antibodies,
T-cell receptor molecules and the like), involvement in cell adhesion
(for example the ICAM molecules) and intracellular signaling (for
example, receptor molecules, such as the PDGF receptor).

[0066] As described herein, an "antigen" is a molecule that is bound by a
binding site on a polypeptide agent. Typically, antigens are bound by
antibody ligands and are capable of raising an antibody response in vivo.
An antigen can be a polypeptide, protein, nucleic acid or other molecule.
In the case of conventional antibodies and fragments thereof, the
antibody binding site as defined by the variable loops (L1, L2, L3 and
H1, H2, H3) is capable of binding to the antigen. The term "antigenic
determinant" refers to an epitope on the antigen recognized by an
antigen-binding molecule (such as bispecific polypeptide agent described
herein), and more particularly, by the antigen-binding site of said
molecule.

[0067] As used herein, an "epitope" can be formed both from contiguous
amino acids, or noncontiguous amino acids juxtaposed by tertiary folding
of a protein. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents, whereas epitopes formed by
tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 3, and more usually, at
least 5, about 9, or about 8-10 amino acids in a unique spatial
conformation. An "epitope" generally includes the unit of structure
conventionally bound by an immunoglobulin VH/VL pair, although
it is recognized that, for example, a single domain antibody may only
require a VH or a VL to recognize and bind to an antigen.
Epitopes define the minimum binding site for an antibody, and thus
represent the target of specificity of an antibody. The terms "antigenic
determinant" and "epitope" can also be used interchangeably herein.

[0068] With respect to a target, the term "ligand interaction site" on the
target means a site, epitope, antigenic determinant, part, domain or
stretch of amino acid residues on the target that is a site for binding
to a ligand, receptor or other binding partner, a catalytic site, a
cleavage site, a site for allosteric interaction, a site involved in
multimerisation (such as homomerization or heterodimerization) of the
target or antigen; or any other site, epitope, antigenic determinant,
part, domain or stretch of amino acid residues on the target or antigen
that is involved in a biological action or mechanism of the target, i.e.,
SGK1. More generally, a "ligand interaction site" can be any site,
epitope, antigenic determinant, part, domain or stretch of amino acid
residues on the SGK1 polypeptide to which an inhibitor or agonist
described herein can bind, such that SGK1 activity and/or expression is
(and/or any pathway, interaction, signalling, biological mechanism or
biological effect in which SGK1 is involved) is modulated.

[0069] An agent (such as a small molecule, an RNA interference molecule,
an antibody, or generally an antigen binding protein or polypeptide or a
fragment thereof) that can specifically bind to, that has affinity for,
and/or that has specificity for a specific antigenic determinant,
epitope, antigen or protein (or for at least one part, fragment or
epitope thereof) is said to be "against" or "directed against" said
antigenic determinant, epitope, antigen or protein.

[0070] An "RNA interference molecule" as used herein, is defined as any
agent which interferes with or inhibits expression of a target gene or
genomic sequence by RNA interference (RNAi). RNA interference involves
the formation and activity of the RNA-induced silencing complex (RISC)
(Gregory R I et al., 2005, Cell 123 (4): 631-640). Such RNA interfering
agents include, but are not limited to, nucleic acid molecules including
RNA molecules which are homologous to the SGK1 target gene or genomic
sequence, or a fragment thereof, short interfering RNA (siRNA), short
hairpin or small hairpin RNA (shRNA), microRNA (miRNA) and small
molecules which interfere with or inhibit expression of the SGK1 target
gene by RNA interference (RNAi).

[0071] The term "screening" as used herein refers to the use of cells,
tissues, or derivatives of them in the laboratory to identify agents with
a specific function, e.g., a modulating activity. In some embodiments,
described herein are screening methods to identify agents (e.g.,
compounds or drugs) that inhibit or otherwise modulate SGK1 activity.

[0072] The term "library," as used herein, refers to a mixture of
heterogeneous agents, such as small molecules, polypeptides or nucleic
acids. The library is composed of members, each of which have a single
small molecule, polypeptide or nucleic acid sequence. To this extent,
library is synonymous with repertoire. Structural and/or sequence
differences between library members are responsible for the diversity
present in the library. The library can take the form of a simple mixture
of small molecules, polypeptides or nucleic acids, or can be in the form
of organisms or cells, for example bacteria, viruses, animal or plant
cells and the like, transformed with a library of, e.g., nucleic acids.
Preferably, each individual organism or cell contains only one or a
limited number of library members. Advantageously, the nucleic acids are
incorporated into expression vectors, in order to allow expression of the
polypeptides encoded by the nucleic acids. Therefore, in some
embodiments, a library can take the form of a population of host
organisms, each organism containing one or more copies of an expression
vector containing a single member of the library in nucleic acid form
which can be expressed to produce its corresponding polypeptide member.
Thus, the population of host organisms has the potential to encode a
large repertoire of genetically diverse polypeptide variants.

[0073] A "marker" as used herein is used to describe the characteristics
and/or phenotype of a cell, e.g., a TH17 cell marker. Markers can be used
for selection of cells comprising characteristics of interests. Markers
will vary with specific cells. Markers are characteristics, whether
morphological, functional or biochemical (enzymatic) characteristics of
the cell of a particular cell type, or molecules expressed by the cell
type. Preferably, such markers are proteins, and more preferably, possess
an epitope for antibodies or other binding molecules available in the
art. However, a marker may consist of any molecule found in or on the
surface of a cell including, but not limited to, proteins (peptides and
polypeptides), lipids, polysaccharides, nucleic acids and steroids.
Examples of morphological characteristics or traits include, but are not
limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of
functional characteristics or traits include, but are not limited to, the
ability to express or produce one or more specific cytokines or
chemokines, the ability to adhere to particular substrates, ability to
incorporate or exclude particular dyes, ability to migrate under
particular conditions, and the ability to differentiate along particular
lineages. Markers may be detected by any method available to one of skill
in the art. Markers can also be the absence of a morphological
characteristic or absence of proteins, lipids etc. Markers can be a
combination of a panel of unique characteristics of the presence and
absence of polypeptides and other morphological characteristics. When a
marker is a protein receptor or other such molecule expressed on the
surface of a cell, it is termed herein as a "cell-surface marker."

[0074] As used herein, an "immune response" refers to a response by a cell
of the immune system, preferably a TH17 cell, but also including a B
cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell,
dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil,
eosinophil, or neutrophil, to a stimulus. In some embodiments, the
response is specific for a particular antigen (an "antigen-specific
response"), and refers to a response by a CD4 T cell, such as a TH17
cell, CD8 T cell, or B cell, via their antigen-specific receptor. In some
embodiments, an immune response is a T cell response, such as a CD4+ TH17
response. Such responses by these cells can include, for example,
cytokine or chemokine production, proliferation, cytotoxicity,
trafficking, or phagocytosis, and can be dependent on the nature of the
immune cell undergoing the response.

[0075] As used herein, "T-cell trafficking" refers to migration of T
lymphocytes, such as the TH17 cells described herein, to a site of an
immune response. Naive T cells recirculate throughout the body, leaving
and reentering the lymphoid tissues as they sample their environment for
the presence of non-self antigens. Lymphoid tissues are specially adapted
to help promote encounters between antigen-specific T-cell receptors
expressed on T cells and their cognate antigens. Specialized
antigen-presenting cells (APCs) concentrate within lymphoid tissues, and
are specially adapted to interact with and to present antigens to T cells
to initiate an immune response by T cells genetically programmed to
recognize a particular antigen. Following T-cell activation in response
to encounter with a specific antigen, T cells proliferate, undergo
differentiation to produce a variety of secreted and cell-associated
products, including cytokines, and migrate to tissue sites associated
with the antigen. The result of this process is that naive T cells
circulate randomly while activated T cells proliferate and home or
traffic to specific tissue sites, i.e., sites of immune responses.

[0078] The term "cytotoxic agent" as used herein refers to a substance
that inhibits or prevents the function of cells and/or causes destruction
of cells. The term is intended to include radioactive isotopes (e.g.
At211, I131, I125, Y90, Re186, Re188,
Sm153, Bi212, P32 and radioactive isotopes of Lu),
chemotherapeutic agents, and toxins such as small molecule toxins or
enzymatically active toxins of bacterial, fungal, plant or animal origin,
including fragments and/or variants thereof.

[0079] As used herein, the terms "chemotherapy" or "chemotherapeutic
agent" refer to any chemical agent with therapeutic usefulness in the
treatment of diseases characterized by abnormal cell growth. Such
diseases include tumors, neoplasms and cancer as well as diseases
characterized by hyperplastic growth. Chemotherapeutic agents as used
herein encompass both chemical and biological agents. These agents
function to inhibit a cellular activity upon which the cancer cell
depends for continued survival. Categories of chemotherapeutic agents
include alkylating/alkaloid agents, antimetabolites, hormones or hormone
analogs, and miscellaneous antineoplastic drugs. Most if not all of these
agents are directly toxic to cancer cells and do not require immune
stimulation. In one embodiment, a chemotherapeutic agent is an agent of
use in treating neoplasms such as solid tumors. In one embodiment, a
chemotherapeutic agent is a radioactive molecule. One of skill in the art
can readily identify a chemotherapeutic agent of use (e.g. see Slapak and
Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles
of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in
Abeloff, Clinical Oncology 2nd ed., COPYRGT. 2000 Churchill
Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to
Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S,
Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed.
St. Louis, Mosby-Year Book, 1993).

[0080] By "radiation therapy" is meant the use of directed gamma rays or
beta rays to induce sufficient damage to a cell so as to limit its
ability to function normally or to destroy the cell altogether. It will
be appreciated that there will be many ways known in the art to determine
the dosage and duration of treatment. Typical treatments are given either
as a one-time treatment or at intervals of e.g., daily, twice a week,
three times a week, weekly, or less frequently as judged by the
administering clinician, and typical dosages range from 10 to 200 units
(Grays) per day.

[0081] As used herein the term "comprising" or "comprises" is used in
reference to compositions, methods, and respective component(s) thereof,
that are essential to the invention, yet open to the inclusion of
unspecified elements, whether essential or not.

[0082] As used herein the term "consisting essentially of" refers to those
elements required for a given embodiment. The term permits the presence
of additional elements that do not materially affect the basic and novel
or functional characteristic(s) of that embodiment of the invention.

[0083] The term "consisting of" refers to compositions, methods, and
respective components thereof as described herein, which are exclusive of
any element not recited in that description of the embodiment.

[0084] As used in this specification and the appended claims, the singular
forms "a," "an," and the include plural references unless the context
clearly dictates otherwise. Thus for example, references to "the method"
includes one or more methods, and/or steps of the type described herein
and/or which will become apparent to those persons skilled in the art
upon reading this disclosure and so forth.

[0085] Other than in the operating examples, or where otherwise indicated,
all numbers expressing quantities of ingredients or reaction conditions
used herein should be understood as modified in all instances by the term
"about." The term "about" when used in connection with percentages can
mean±1%.

[0086] Unless otherwise defined herein, scientific and technical terms
used in connection with the present application shall have the meanings
that are commonly understood by those of ordinary skill in the art to
which this disclosure belongs. It should be understood that this
invention is not limited to the particular methodology, protocols, and
reagents, etc., described herein and as such can vary. The terminology
used herein is for the purpose of describing particular embodiments only,
and is not intended to limit the scope of the present invention, which is
defined solely by the claims. Definitions of common terms in immunology,
and molecular biology can be found in The Merck Manual of Diagnosis and
Therapy, 18th Edition, published by Merck Research Laboratories, 2006
(ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopedia of
Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN
0-632-02182-9); and Robert A. Meyers (ed.),

[0088] It is understood that the foregoing detailed description and the
following examples are illustrative only and are not to be taken as
limitations upon the scope of the invention. Various changes and
modifications to the disclosed embodiments, which will be apparent to
those of skill in the art, may be made without departing from the spirit
and scope of the present invention. Further, all patents, patent
applications, and publications identified are expressly incorporated
herein by reference for the purpose of describing and disclosing, for
example, the methodologies described in such publications that might be
used in connection with the present invention. These publications are
provided solely for their disclosure prior to the filing date of the
present application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such disclosure
by virtue of prior invention or for any other reason. All statements as
to the date or representation as to the contents of these documents are
based on the information available to the applicants and do not
constitute any admission as to the correctness of the dates or contents
of these documents.

BRIEF DESCRIPTION OF THE FIGURES

[0089] FIG. 1 shows that TGF-β can upregulate the expression level of
SGK1. FIG. 1 further demonstrates that addition of IL-6 together with
TGF-β further increased the expression SGK1. IL-23, an IL-12 family
cytokine, essential for enhancing generation of TH17 cells was found to
further enhance the expression of SGK1.

[0090] FIG. 2 shows time-dependent expression of SGK1 during TH17
differentiation. Naive CD4+ T cells were cultured in TH17 differentiating
condition (TGF-β plus IL-6) for 96 hours. After 48 hours, TH17 cells
were supplemented with IL-23 until the end of the culture, and it was
found that expression of SGK1 was rapidly induced after 2 hours and
dropped down to the base level after 8 hours, and that IL-23 further
induced SGK1 expression.

[0092] FIG. 4 shows that SGK1 is essential for IL-23 dependent expansion
of TH17 cells. Naive CD4+ T cells were sorted from wild-type and
SGK1-deficient mice and cultured under TH17 differentiation conditions.
After a first round of stimulation, the cells were rested for two days in
cytokine free medium. Two days later, cells were activated in the
presence or absence of IL-23, and intracellular cytokine staining was
performed for IL-17. IL-23 was able to expand already differentiated
wild-type TH17 cells (11% to 13%), however SGK1-deficient TH17 cells
failed to undergo IL-23 mediated expansion (˜4% to ˜1%).

[0093]FIG. 5 demonstrates that SGK1 is essential for IL-23 mediated
expansion of TH17 cells. Wild-type and SGK1-deficient CD4+CD62L-cells
(memory CD4 cells) were sorted and cultured with either anti-CD3 alone or
anti-CD3 plus IL-23. IL-23 clearly enhanced the expression of IL-17A and
IL-17F in wild-type cells, however SGK1-deficient memory cells were
defective in inducing expression of IL-17A and IL-17F.

[0094]FIG. 6 demonstrates that inhibitors of PI3 kinase and AKT/MAP
kinase do not influence SGK1 upregulation mediated by TGF-β and
IL-6.

DETAILED DESCRIPTION

[0095] Described herein are novel compositions and methods for modulating
TH17 cell differentiation and activity via inhibition or activation of
SGK1. These compositions and methods are based, in part, on the novel
discovery by the inventors that SGK1, a serine/threonine kinase
previously described as being involved in regulation of cellular sodium
homeostasis, has a novel and unexpected function in the differentiation
and function of a specific subset of CD4 T cells, the TH17 lineage, while
not impacting the differentiation and function of other subsets of CD4 T
cells, such as TH1 or TH2 cells. Accordingly, described herein are
methods and compositions for modulation of TH17 cell differentiation,
proliferation, activity, and/or function that rely upon modulating the
activity or expression of SGK1. Such methods and compositions are useful
in the treatment of a variety of disorders including autoimmune diseases,
chronic inflammatory conditions, infectious diseases, and cancer.

TH17 Cells

[0096] Distinct types of adaptive immune responses affording protection
against different classes of pathogens are facilitated by the
differentiation of CD4+ T cells into the corresponding types of effector
T cells, which currently comprise TH1, TH2, and TH17 subsets. Through
elaboration of distinct sets of cytokines and other soluble and
cell-bound products, these cells act as immune effectors eliminating
cells infected by pathogens. Importantly, such differentiated CD4+ T
cells act as principal amplifiers and inducers of the appropriate
inflammatory and effector responses in cells of the innate immune system
and "nonimmune" cells. The amplified blocks of adaptive and innate immune
responses lead to efficient clearance or containment of offending
pathogens.

[0097] The downside of powerful mechanisms of protection against pathogen
afforded by the immune system of higher organisms is inflammation
associated with the "unwanted" immune responses against "self", i.e., in
autoimmune disorders, and environmental antigens and commensal
microorganisms, i.e., in allergic and atopic disorders, as well as
"collateral" damage to the host as a side effect of immune responses
against pathogens. These side effects can be, at times, more devastating
than the infection itself. TH17 cells have been implicated in numerous
autoimmune diseases and other inflammatory conditions, and are most
abundant at mucosal surfaces, particularly the intestinal lamina propria
(LP).

[0098] Following infection with diverse microbes, T cells undergo
differentiation when their TCRs are triggered in the presence of
particular combinations of cytokines produced by innate immune cells
([Abbas et al., 1996] and [Mosmann and Coffman, 1989]). Infection of
myeloid cells with intracellular bacteria and viruses typically elicits
production of IL-12, which induces differentiation of interferon-γ
(IFN-γ)-producing Th1 cells and cytotoxic CD8+ T cells that
are best suited to clear such pathogens. Infection with parasitic worms,
in contrast, induces production of IL-4 by cells of the innate immune
system, and this, in turn, stimulates CD4+ T cells to differentiate
into TH2 cells that produce more IL-4, as well as IL-5 and IL-13,
cytokines involved in controlling expulsion of the helminths.

[0099] The third, recently described, subset of CD4 T helper cells, TH17
cells, are abundant at mucosal interfaces, where they contain infection
with pathogenic bacteria and fungi (Weaver et al., 2007). These cells
produce IL-17A (also referred to as IL-17), IL-17F, and IL-22, cytokines
involved in neutrophilia, tissue remodeling and repair, and production of
antimicrobial proteins.

[0100] TH17 cells differentiate in response to the STAT3-activating
cytokines IL-6, IL-21, and IL-23, along with TGF-β and IL-1β
(Korn et al., 2009). TH17 cells can also further comprise subsets of
cells that produce IL-22, but not IL-17, such as skin-homing T helper
cells that produce IL-22, but not IL-17 (Duhen et al., 2009). The
differentiation of CD4+ T cells that produce IL-17 and IL-22 is
influenced by the composition of the intestinal microbiota and by the
presence of innate immune cells that amplify the TH17 cell response.

[0101] TH17 cells have been shown to differentiate in vitro from naive
CD4+ T cells in response to TCR signaling in the presence of IL-6
and TGF-β, but not IL-23 (Bettelli et al., 2006 and Veldhoen et al.,
2006). The receptor for IL-23 is expressed on naive murine CD4+ T
cells only after stimulation in the presence of IL-6 or IL-21, and then
these other cytokines can give way to the ability of IL-23 to stimulate
continued differentiation of TH17 cells and, perhaps, their survival
(Korn et al., 2007, Nurieva et al., 2007 and Zhou et al., 2007). In human
T cells, IL-23R can be constitutively expressed on CD4+ T cells, and
hence IL-17 expression can be induced by IL-23 in vitro (Manel et al.,
2008). Mouse T cells bearing γδ TCRs, which are prominent in
mucosal tissues, also express IL-23R constitutively and have been
reported to differentiate into IL-17-producing cells early after exposure
to IL-23 (Roark et al., 2008).

[0102] The "TH17 program," as used herein refers to the expression of
signature cytokines, the chemokine receptor CCR6, and IL-23R by a CD4+
TH17 cell. Some of these same features can also be found in other
lymphoid cells, e.g., TCRγδ T cells, lymphoid tissue inducer
(LTi) cells, and phenotypically related cells with NK cell markers, that
secrete IL-17 and/or IL-22 (Colonna, 2009). These cells share with CD4+
TH17 cells the expression of the orphan nuclear receptor RORγt,
which is both necessary and sufficient for expression of the genes that
currently define the TH17 program (Ivanov et al., 2006). In addition to
RORγt, other transcription factors have been shown to be required
for the expression of IL-17 in polarized T helper cells, and several of
these are also required for upregulation of RORγt upon
polarization. These include IRF4 and BATF, whose expression is induced
upon TCR signaling, and STAT3 (Brustle et al., 2007, Schraml et al.,
2009, and Zhou and Littman, 2009). Additional transcription factors that
contribute to the induction of IL-17 in polarized cells include, but are
not limited to, Runx1/CBFβ, c-Maf, and the ligand-regulated aryl
hydrocarbon receptor (AhR) (Bauquet et al., 2009, Veldhoen et al., 2008
and Zhang et al., 2008). RORα also contributes to some IL-17
expression in the absence of RORγt (Yang et al., 2008b). AhR has
been shown to be required for induction of IL-22 in response to
xenobiotic ligands. STAT3, IRF4, and BATF are required for expression of
RORγt in TH17-polarized T helper cells, yet each contributes
additionally, in cooperation with RORγt, to expression of IL-17
and, other key components of the TH17 program.

SGK1

[0103] SGK1 (or serum/glucocorticoid-regulated kinase 1 or
serine/threonine-protein kinase 1) is a serine/threonine protein kinase
that has been shown to play an important role in cellular stress
responses. This kinase activates certain potassium, sodium, and chloride
channels, and is involved in the regulation of processes such as cell
survival, neuronal excitability, and renal sodium excretion. High levels
of expression of this gene have been thought to contribute to conditions
such as hypertension and diabetic nephropathy. Several alternatively
spliced transcript variants encoding different isoforms have been
described for SGK1.

[0104] Accordingly, the term "SGK1" as used herein, refers to any of the
following naturally occurring SGK1 isoforms having the amino acid
sequence of:

[0105] SGK1 was described for the first time in 1993 as an immediate early
gene in a rat mammary carcinoma cell line (Webster et al., 1993a; Webster
et al., 1993b). It was shown in further studies that SGK-1 and its
inducibility occurs in various cell lines and in cells of normal tissues
(Brennan et al., 2000; Naray-Fejes-Toth et al., 2000; Cooper et al.,
2001; Mikosz et al., 2001). SGK-1 belongs to a family of serine/threonine
kinases of which to date three members are known and are referred to as
SGK-1, SGK-2 and SGK-3/SGKL/CISK.

[0108] Described herein are novel therapeutic agents and methods for
modulating TH17-mediated immune responses by inhibiting or activating
SGK1 expression and/or activity. These therapeutic agents and uses
thereof, and methods of modulating TH17 responses are based, in part, on
the inventors' surprising discovery that the serine/threonine kinase
SGK1, which had previously been primarily implicated in sodium regulation
and blood pressure maintenance, has novel roles in the differentiation
and maintenance of CD4+ TH17 cells. The inventors have discovered
that in the absence of SGK1 expression, de novo TH17 differention from
naive CD4+ T cells in the presence of TGF-β and IL-6 is
significantly impaired. Further, the inventors have found that the
survival, maintenance, and stability of TH17 cells in the presence of
IL-23 is significantly impaired in the absence of SGK1 expression.
Furthermore, the inventors found that SGK1 expression is specific to
regulation of TH17 cells, as no changes in Th1 and TH2 cell
differentiation was observed in the absence of SGK1. Thus, SGK1
represents a novel target for specifically modulating TH17 responses.

[0109] Accordingly, described herein are therapeutic compositions, and
methods of use thereof, for inhibiting SGK1 expression for the treatment
of disorders mediated by dysregulated or increased TH17 cell activity,
such as in the treatment of autoimmune disorders and other
proinflammatory disorders. In other aspects, described herein are
therapeutic compositions, and methods of use thereof, for activating or
increasing SGK1 expression for the treatment of disorders in which
increased TH17 cell activity and function provides therapeutic benefits,
such as in infectious disorders.

[0110] Described herein are modulators of SGK1 activity and their use for
the treatment of disorders and disease conditions whereby modulation of
TH17 cell activity and/or function has beneficial effects and outcomes.
Such SGK1 modulators include agents such as small molecules, nucleic
acids, polypeptides, peptides, drugs, etc. An "SGK1 modulating agent"
refers to any chemical, entity, or moiety, including without limitation
synthetic and naturally-occurring proteinaceous (e.g., antibodies or
antigen-binding fragments thereof) and non-proteinaceous (e.g., small
molecule or nucleic acid-based) entities, that causes or facilitates a
qualitative or quantitative change, alteration, or modification in one or
more processes, mechanisms, effects, responses, functions, activities or
pathways mediated by SGK1. Such changes mediated by an SGK1 modulating
agent, such as an SGK1 inhibitor or an SGK1 activating agent described
herein, can refer to a decrease or an increase in the activity or
function of SGK1, such as a decrease or increase in, or inhibition or
activation of, serine/threonine phosphorylation activity of SGK1, where,
e.g., SGK1 enzymatic activity is assayed as described herein. Such
modulating can, for example, also involve allosteric modulation of SGK1;
and/or reducing or inhibiting the binding of SGK1 to one of its
substrates or ligands, and/or competing with a natural ligand or
substrate for binding to SGK1. Modulating can also involve activating the
target or antigen or the mechanism or pathway in which it is involved. An
SGK1 modulating agent can, for example, also cause or effect a change in
respect to the folding or conformation of SGK1 (for example, upon binding
of a ligand or interaction with a substrate), to associate with other
(sub)units, or to disassociate from one or more subunits, or from a
complex, such as an enzyme complex.

[0111] In some embodiments of the aspects described herein, an SGK1
modulating agent is a nucleic acid, a nucleic acid analogue, protein,
antibody, peptide, aptamer, oligomer of nucleic acids, amino acid, or
carbohydrate, and includes, without limitation, proteins,
oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs,
lipoproteins, aptamers, and modifications and combinations thereof. SGK1
agonists, activators, inhibitors, or antagonists can be naturally
occurring and as a group, comprises synthetic ligands, small chemical
molecules, antibodies or antigen-binding fragments thereof, polypeptides
(e.g., dominant-negative SGK1 polypeptides), inhibitory RNA molecules
(i.e., siRNA or antisense RNA), and the like. Such SGK1 modulating agents
can be selected from compounds known to have a desired activity and/or
property, or can be selected from a library of diverse compounds by
screening methods, as known to one of skill in the art. For example,
assays to identify SGK1 inhibitors and SGK1 activators include, e.g.,
applying or contacting putative SGK1 modulator compounds to cells, in the
presence or absence of an SGK1 polypeptide or polynucleotide encoding
SGK1, and then determining changes in expression or functional effects of
the putative SGK1 modulator compound on the SGK1 polypeptide or
polynucleotide encoding SGK1.

[0112] A variety of assays can be used to assay for SGK1 expression and/or
activity. Transcript (mRNA) expression of SGK1 can be ascertained by any
standard method known to one of skill in the art, such as Northern blot
analysis, semi- or real-time quantitative PCR analyses, and nucleic
acid-based high-throughput chip assays, such as microarrays. Protein
expression of SGK1 can be determined using any standard method known to
one of skill in the art, such as Western blot analysis, flow cytometric
assays for intracellular molecules, kinase substrate assays, and the
like.

[0113] The ability of an SGK1 modulating agent, i.e., SGK1 inhibitor or
SGK1 activator, such as a small molecule or antibody or antigen-binding
fragment thereof, to modulate SGK1 activity, such as kinase activity, can
be ascertained using any of a variety of assays known to one of skill in
the art. These include a variety of kinase assays known in the literature
that can be readily performed by one of skill in the art as described in,
for example, Dhanabal et al., Cancer Res. 59:189-197; Xin et al., J.
Biol. Chem. 274:9116-9121; Sheu et al., Anticancer Res. 18:4435-4441;
Ausprunk et al., Dev. Biol. 38:237-248; Gimbrone et al., J. Natl. Cancer
Inst 52:413-427; Nicosia et al., In vitro 18:538-549.

[0114] For example, SGK1 or a fragment thereof comprising the kinase
domain, can be expressed for the purposes of protein production in cells,
such as insect cells, e.g., Sf21; S. frugiperda, and subsequently
purified by affinity chromatography as a fusion protein with glutathione
S-transferase in a baculovirus expression vector. The cultivation,
infection and digestion of the cells as well as the purification of the
fusion protein by column chromatography are carried out in accordance
with manufacturer-oriented generic working instructions. Kinase activity
is measured using various available measurement systems. In the
scintillation proximity method (Sorg et al., J. of. Biomolecular
Screening, 2002, 7, 11-19), the flashplate method or the filter binding
test, the radioactive phosphorylation of a protein or peptide as
substrate is measured using radioactively labelled ATP (32P-ATP,
33P-ATP). In the case of the presence of an inhibitory compound, a
reduced radioactive signal, or none at all, can be detected. Furthermore,
homogeneous time-resolved fluorescence resonance energy transfer
(HTR-FRET) and fluorescence polarisation (FP) technologies, are useful as
assay methods (Sills et al., J. of Biomolecular Screening, 2002,
191-214). Other non-radioactive ELISA assay methods use specific
phospho-antibodies (phospho-ABs). Such phospho-antibodies only bind the
phosphorylated substrate. Such binding can be detected by
chemiluminescence using a second peroxidase-conjugated antibody (Ross et
al., 2002, Biochem. J.). In another example, fluorescence polarization
can be used to monitor binding of a putative SGK1 modulator to SGK1
and/or monitor SGK1 kinase activity.

[0115] In some aspects, the SGK modulating agents described herein are
SGK1 inhibitors. As used herein, the terms "inhibitor of SGK1" or "SGK1
inhibitor" refer to an agent or compound that inhibits SGK1 signaling and
downstream effector pathways, as those terms are used herein. In some
embodiments of the aspects described herein, the downstream effector
pathway inhibited by the SGK1 inhibitor is TH17 cell differentiation or a
TH17 cell-activity mediated by SGK1 expression, and thus the SGK1
inhibitor is an inhibitor of TH17 cell differentiation or TH17
cell-activity. Thus, the term SGK1 inhibitor refers to an agent that:
inhibits expression of an SGK1 polypeptide, including any of the
polypeptides of SEQ ID NO:1-SEQ ID NO:4; inhibits expression of a
polynucleotide encoding SGK1, including any polynucleotide sequence
encoding any of the polynucleotides of SEQ ID NO:1-SEQ ID NO:4; or one
that binds to, partially or totally blocks stimulation of, decreases,
prevents, delays activation of, inactivates, desensitizes, or
downregulates the activity of an SGK1 polypeptide or polynucleotide
encoding SGK1. Such SGK1 inhibitors can e.g., inhibit SGK1 expression,
e.g., SGK1 translation, post-translational processing of SGK1, stability,
degradation, or nuclear or cytoplasmic localization of an SGK1
polypeptide, or transcription, post transcriptional processing, stability
or degradation of a polynucleotide encoding SGK1, or bind to, partially
or totally block stimulation of, or enzymatic (e.g., kinase) activity of
SGK1. An SGK1 inhibitor can act directly or indirectly. SGK1 inhibition
is achieved when the activity value of an SGK1 polypeptide, or a
polynucleotide encoding SGK1 is about at least 10% less, and preferably,
at least 20% less, at least 30% less, at least 40% less, at least 50%
less, at least 60% less, at least 70% less, at least 80% less, at least
90% less, at least 95% less, or absent or undetectable in comparison to a
reference or control level in the absence of the SGK1 inhibitor.

[0116] In some embodiments of these aspects, the SGK1 inhibitor is an
antagonist. An SGK1 antagonist refers to an SGK1 inhibitor that does not
provoke a biological response itself upon specifically binding to an SGK1
polypeptide or polynucleotide encoding SGK1, but blocks or dampens
agonist-mediated or ligand-mediated responses, i.e., an SGK1 antagonist
can bind, but does not activate, an SGK1 polypeptide or polynucleotide
encoding SGK1, and the binding disrupts the interaction with an
endogenous or exogenous SGK1 substrate, ligand, or agonist, displaces an
endogenous or exogenous SGK1 substrate, ligand, or agonist, and/or
inhibits the function of an SGK1 substrate, ligand, or agonist. SGK1
antagonists can mediate their effects by binding to, for example, the
active site, i.e., enzymatic site, or to allosteric sites on an SGK1
polypeptide or a polynucleotide encoding SGK1.

[0117] In some embodiments of the aspects described herein, an SGK1
inhibitor is a small molecule of Formula (I):

##STR00028##

wherein R1 is optionally substituted phenyl, optionally substituted
β-napthyl, or optionally substituted 3-CN-phenyl; wherein R2 is
CO2R4 or C(R4,R5) CO2R4; wherein R3 and R4 are independently
absent, H, C1-C6 alkyl, or C5-C8 cycloalkyl; each of
which may be optionally substituted; wherein R5 and R6 are independently
absent, H, or C1-C6 alkyl, each of which may be optionally
substituted; and pharmaceutically acceptable salts thereof.

[0118] In some embodiments, the SGK1 inhibitor of Formula (I), is a small
molecule of Formula (Ia):

##STR00029##

[0119] In some embodiments of Formula (Ia), R1 is phenyl, R2 is CO2H,
and R3 is H. In some embodiments, R1 is phenyl, R2 is CO2, and R3 is

##STR00030##

In some embodiments, R1 is phenyl, R2 is CO2H, and R3 is

##STR00031##

In some embodiments, R1 is phenyl, R2 is CO2H, and R3 is

##STR00032##

In some embodiments, R1 is β-napthyl, R2 is CH2CO2H, and
R3 is H. In some embodiments, R1 is β-napthyl, R2 is

[0121] In other embodiments of these aspects, the small molecule
inhibitors of SGK1 can include, but are not limited to, the SGK1
antagonist GSK650394
(2-Cyclopentyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-benzoic acid) or
derivatives thereof, described in Cancer Res 2008; 68(18):7475-83, the
contents of which are herein incorporated in their entirety by reference;
the small molecule indazolesquaric acid derivatives described in U.S.
Patent Publication No.: US2009/0036449, the contents of which are herein
incorporated in their entirety by reference; and the Acylhydrazone
derivative and Pyridopyrimidine derivative SGK1 inhibitors described in
U.S. Patent Publication No.: US2007/0191326 or in WO/2007/121963, the
contents of which are herein incorporated in their entirety by reference.
Other compounds of Formula (Ia) are described in WO2006/063167 and
Bioorg. Med. Chem. Lett. 2009, 19, 4441-4445, the contents of which are
herein incorporated in their entirety by reference.

[0122] For simplicity, chemical moieties as defined and referred to
throughout can be univalent chemical moieties (e.g., alkyl, aryl, etc.)
or multivalent moieties under the appropriate structural circumstances
clear to those skilled in the art. For example, in some embodiments, an
"alkyl" moiety can refer to a monovalent radical (e.g.
CH3--CH2--), or in other embodiments, a bivalent linking moiety
can be "alkyl," in which case those skilled in the art will understand
the alkyl to be a divalent radical (e.g., --CH2--CH2--), which
is equivalent to the term "alkylene." Similarly, in circumstances in
which divalent moieties are required and are stated as being "alkoxy",
"alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl",
"heterocyclic", "alkyl" "alkenyl", "alkynyl", "aliphatic", or
"cycloalkyl", those skilled in the art will understand that the terms
"alkoxy", "alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl",
"heterocyclic", "alkyl", "alkenyl", "alkynyl", "aliphatic", or
"cycloalkyl" refer to the corresponding divalent moiety.

[0123] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine.

[0124] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl,
arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,
any of which may be further substituted by substituents. Exemplary acyl
groups include, but are not limited to, (C1-C6)alkanoyl (e.g.,
formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl,
etc.), (C3-C6)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,
cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),
heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,
pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl,
tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl
(e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl,
furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,
benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl, cycloalkyl,
heterocycle, aryl and heteroaryl portion of the acyl group may be any one
of the groups described in the respective definitions.

[0125] The term "alkyl" refers to saturated non-aromatic hydrocarbon
chains that may be a straight chain or branched chain, containing the
indicated number of carbon atoms (these include without limitation
methyl, ethyl, propyl, butyl, pentyl, hexanyl, which may be optionally
inserted with N, O, S, SS, SO2, C(O), C(O)O, OC(O), C(O)N or NC(O).
For example, C1-C6 indicates that the group may have from 1 to
6 (inclusive) carbon atoms in it.

[0126] The term "alkenyl" refers to an alkyl that comprises at least one
double bond. Exemplary alkenyl groups include, but are not limited to,
for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl and the
like.

[0127] The term "alkynyl" refers to an alkyl that comprises at least one
triple bond.

[0128] The term "alkoxy" refers to an --O-alkyl radical.

[0129] The term "aminoalkyl" refers to an alkyl substituted with an amino.

[0130] The term "aryl" refers to monocyclic, bicyclic, or tricyclic
aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted by a substituent. Exemplary aryl groups include, but are not
limited to, phenyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl,
indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.

[0131] The term "arylalkyl" refers to alkyl substituted with an aryl or
aryl substituted with an alkyl.

[0132] The term "cycloalkyl" refers to saturated and partially unsaturated
cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8
carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group
additionally may be optionally substituted. Exemplary cycloalkyl groups
include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and the
like.

[0133] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring
system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic,
or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or
S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if
monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or
4 atoms of each ring may be substituted by a substituent. Exemplary
heteroaryl groups include, but are not limited to, pyridyl, furyl or
furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl,
pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl,
and the like.

[0134] The term "heteroarylalkyl" refers to an alkyl substituted with a
heteroaryl.

[0135] The term "heterocyclyl" refers to a non-aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring
system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic,
or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or
S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if
monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3
atoms of each ring may be substituted by a substituent. Exemplary
heterocyclyl groups include, but are not limited to piperazinyl,
pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

[0136] The term "haloalkyl" refers to an alkyl group having one, two,
three or more halogen atoms attached thereto. Exemplary haloalkyl groups
include, but are not limited to chloromethyl, bromoethyl,
trifluoromethyl, and the like.

[0137] The term "optionally substituted" means that the specified group or
moiety, such is unsubstituted or is substituted with one or more
(typically 1-4 substituents) independently selected from the group of
substituents listed herein in the definition for "substituents" or
otherwise specified. The substituents may be "separate" substituents, for
instance, a halo group and an alkoxy groups bonded to different carbon
atoms in a benzene ring, or the substituents may be "stacked" on one
another, for instance, an acyl group (such as formyl) that is substituted
with an aminosulfonyl group that is substituted with an arylalkyl (such
as toluene).

[0138] The term "substituents" refers to a group that replaces a hydrogen
at any atom of the substituted group or moiety, as well as a group
"substituted" on an alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heterocyclyl, heteroaryl, acyl, amino group at any atom of that group.
Suitable substituents include, without limitation, halo, hydroxy, oxo,
nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, aralkyl,
alkoxy, aryloxy, amino, aminosulfonyl, acylamino, alkylcarbanoyl,
arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,
alkylthio, CF3, N-morphilino, phenylthio, alkanesulfonyl,
arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,
alkylcarbonyl, acyloxy, cyano or ureido. In some cases, two substituents,
together with the carbons to which they are attached to can form a ring.

[0139] In many cases, protecting groups are used during preparation of the
compounds of the invention. As used herein, the term "protected" means
that the indicated moiety has a protecting group appended thereon. In
some preferred embodiments of the invention, compounds contain one or
more protecting groups. A wide variety of protecting groups can be
employed in the methods of the invention. In general, protecting groups
render chemical functionalities inert to specific reaction conditions,
and can be appended to and removed from such functionalities in a
molecule without substantially damaging the remainder of the molecule.

[0141] Nitrogen- or amino-protecting groups stable to acid treatment are
selectively removed with base treatment, and are used to make reactive
amino groups selectively available for substitution. Exemplary
amino-protecting groups include, but are not limited to, carbamate
protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc),
1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC),
allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and
benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl, acetyl,
trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting
groups, such as 2-nitrobenzenesulfonyl; and imine and cyclic imide
protecting groups, such as phthalimido and dithiasuccinoyl.

[0143] In other embodiments, an SGK1 inhibitor comprises an anti-SGK1
antibody or an antigen-binding fragment thereof. When SGK1-specific
antibodies or antigen-binding fragments thereof are used in inhibiting
SGK1 activity and/or expression, it is understood that the antibody or
antigen-binding fragment thereof is a "blocking" antibody or an antibody
"antagonist," i.e., it is one that inhibits or reduces biological
activity of SGK1 upon binding, and does not activate or promote SGK1
signaling. For example, an SGK1 antagonist antibody can bind SGK1 and
inhibit the ability of SGK1 to, for example, phosphorylate serine or
threonine on a enzymatic substrate. In certain embodiments, the blocking
antibodies or antagonist antibodies or fragments thereof described herein
completely inhibit the biological activity of SGK1. Such anti-SGK1
antibodies include all such classes, subclasses and types of human
antibody species. For example, as used herein, antibodies to SGK1
polypeptides also include antibodies to fusion proteins comprising SGK1
polypeptides or fragments of SGK1 polypeptides. More specifically, the
SGK1 inhibitor can be a monoclonal or single specificity anti-SGK1
antibody or antigen-binding fragment thereof. The anti-SGK1 antibody or
antigen-binding fragment thereof may be human, humanized, chimeric, or an
in vitro generated antibody to human SGK1, as described herein. In
addition, anti-SGK1 antibodies are available commercially, e.g., from R&D
Systems, Abcam, and Santa Cruz Biotechnology, Inc.

[0144] Accordingly, in some embodiments of these aspects, the anti-SGK1
blocking antibody or antigen-binding fragment thereof is a human
SGK1-specific antibody fragment. In some embodiments, the anti-SGK1
blocking antibody fragment is a Fab fragment comprising VL, CL,
VH and CH1 domains. In some embodiments, the anti-SGK1 blocking
antibody or antigen-binding fragment thereof is a Fab' fragment, which is
a Fab fragment having one or more cysteine residues at the C-terminus of
the CH1 domain. In some embodiments, the anti-SGK1 blocking antibody
or antigen-binding fragment thereof is a Fd fragment comprising VH
and CH1 domains. In some embodiments, the anti-SGK1 blocking
antibody or antigen-binding fragment thereof is a Fd' fragment comprising
VH and CH1 domains and one or more cysteine residues at the
C-terminus of the CH1 domain. In some embodiments, the anti-SGK1
blocking antibody or antigen-binding fragment thereof is a Fv fragment
comprising the VL and VH domains of a single arm of an
antibody. In some embodiments, the anti-SGK1 blocking antibody or
antigen-binding fragment thereof is a dAb fragment comprising a VH
domain or a VL domain. In some embodiments, the anti-SGK1 blocking
antibody or antigen-binding fragment thereof comprises isolated CDR
regions. In some embodiments, the anti-SGK1 blocking antibody or
antigen-binding fragment thereof is a F(ab')2 fragment, which
comprises a bivalent fragment comprising two Fab' fragments linked by a
disulphide bridge at the hinge region. In some embodiments, the anti-SGK1
blocking antibody or antigen-binding fragment thereof is a single chain
antibody molecule, such as a single chain Fv. In some embodiments, the
anti-SGK1 blocking antibody or antigen-binding fragment thereof is a
diabody comprising two antigen binding sites, comprising a heavy chain
variable domain (VH) connected to a light chain variable domain
(VL) in the same polypeptide chain. In some embodiments, the
anti-SGK1 blocking antibody or antigen-binding fragment thereof is a
linear antibody comprising a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary
light chain polypeptides, form a pair of antigen binding regions.

[0145] In other aspects, the SGK modulating agents described herein are
SGK1 activators. As used herein, the terms "SGK1 activator," "activator
of SGK1," and "SGK1 agonist" refer to an agent that binds to an SGK1
polypeptide or polynucleotide encoding SGK1, and stimulates, increases or
upregulates expression of, or enhances enzymatic (serine/threonine
kinase) activity of an SGK1 polypeptide or polynucleotide encoding SGK1.
An increase in SGK1 activity or SGK1 expression is achieved by an SGK1
activator when the activity of or expression of an SGK1 polypeptide or a
polynucleotide encoding SGK1 is at least 10% higher, at least 20% higher,
at least 30% higher, at least 40% higher, at least 50% higher, at least
60% higher, at least 70% higher, at least 80% higher, at least 90%, at
least 100% higher, at least 2-fold higher, at least 3-fold higher, at
least 5-fold higher, at least 10-fold higher, at least 15-fold higher, at
least 25-fold higher, at least 50-fold higher, at least 100-fold higher,
at least 1000-fold higher, or more, relative to a reference activity or
expression of an SGK1 polypeptide or polynucleotide encoding SGK1 in the
absence of the SGK1 activator.

[0146] In some embodiments of these aspects, the SGK1 activator or agonist
is an antibody or antigen-binding fragment thereof, a polypeptide, a
small molecule, or an activating nucleic acid molecule, such as an
activating RNA molecule.

[0147] When SGK1-specific antibodies or antigen-binding fragments thereof
are used in activating SGK1 activity and/or expression, it is understood
that the antibody or antigen-binding fragment thereof is an "activating"
antibody or an antibody "agonist," i.e., it is one that increases or
promotes biological activity of SGK1, such as promoting TH17
differentiation or TH17 cell activity upon binding. For example, an SGK1
activating antibody can bind SGK1 and promote or increase the ability of
SGK1 to, for example, phosphorylate serine or threonine on an enzymatic
substrate. Such anti-SGK1 activating antibodies include all such classes,
subclasses and types of human antibody species. Such activating
antibodies to SGK1 polypeptides also include antibodies to fusion
proteins comprising SGK1 polypeptides or fragments of SGK1 polypeptides.
The SGK1 activating antibody can be a monoclonal or single specificity
anti-SGK1 antibody or antigen-binding fragment thereof. The anti-SGK1
activating antibody or antigen-binding fragment thereof may be human,
humanized, chimeric, or an in vitro generated activating antibody to
human SGK1, as described herein.

[0148] Accordingly, in some embodiments of these aspects, the anti-SGK1
activating antibody or antigen-binding fragment thereof is a human
SGK1-specific antibody fragment. Activating antibodies or antigen-binding
fragments thereof can take any of the forms for antibodies or
antigen-binding fragments thereof described herein in the context of
antagonist or inhibitory antibodies.

[0149] In some embodiments, an SGK1 activator indirectly activates SGK1
via, for example, activation of an upstream regulator of SGK1, such as
PPARγ. Thus, in some embodiments, an SGK1 activator is a
PPARγ agonist, such as pioglitazone and L-805645.

Production of Anti-SGK1 Antibodies and Antigen-Binding Fragments Thereof

[0150] Non-limiting methods of producing the anti-SGK1 blocking and
agonist antibodies for use in the compositions and methods described
herein are detailed below.

[0151] Polyclonal Antibodies.

[0152] Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous (sc) or intraperitoneal (ip) injections of the relevant
antigen, e.g., SGK1, and an adjuvant. It can be useful, in some
embodiments, to conjugate the relevant antigen to a protein that is
immunogenic in the species to be immunized, e.g., keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin
inhibitor using a bifunctional or derivatizing agent, for example,
maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine
residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride, SOCl2, or R1N═C═NR,
where R and R1 are different alkyl groups.

[0153] Monoclonal Antibodies.

[0154] Various methods for making monoclonal antibodies specific for SGK1
as described herein are available in the art. For example, the monoclonal
antibodies can be made using the hybridoma method first described by
Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods
(U.S. Pat. No. 4,816,567).

[0155] In the hybridoma method, a mouse or other appropriate host animal,
is immunized to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the SGK1 protein or
fragment thereof used for immunization. Alternatively, lymphocytes can be
immunized in vitro. Lymphocytes then are fused with myeloma cells using a
suitable fusing agent, such as polyethylene glycol, to form a hybridoma
cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)). The hybridoma cells thus prepared are seeded and
grown in a suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused, parental
myeloma cells. Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the production of human monoclonal antibodies (Kozbor,
J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,
New York, 1987)). Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
After hybridoma cells are identified that produce antibodies of the
desired specificity, affinity, and/or activity, the clones can be
subcloned by limiting dilution procedures and grown by standard methods
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)). Suitable culture media for this purpose include,
for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells
can be grown in vivo as ascites tumors in an animal. DNA encoding the
monoclonal antibodies can be readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding specifically to genes encoding the heavy and light
chains of the monoclonal antibodies). The hybridoma cells serve as a
preferred source of such DNA. Once isolated, the DNA can be placed into
expression vectors, which are then transfected into host cells such as E.
coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal antibodies in the recombinant host
cells.

[0157] The DNA sequences encoding the antibodies or antibody fragment that
specifically bind SGK1 also can be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant domains in
place of the homologous murine sequences (U.S. Pat. No. 4,816,567;
Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by
covalently joining to the immunoglobulin coding sequence all or part of
the coding sequence for a non-immunoglobulin polypeptide.

[0158] Such non-immunoglobulin polypeptides can be substituted for the
constant domains of an antibody, or they can be substituted for the
variable domains of one antigen-combining site of an antibody to create a
chimeric bivalent antibody comprising one antigen-combining site having
specificity for an antigen and another antigen-combining site having
specificity for a different antigen.

[0159] Humanized and Human Antibodies.

[0160] A humanized antibody has one or more amino acid residues introduced
into it from a source which is non-human. These non-human amino acid
residues are often referred to as "import" residues, which are typically
taken from an "import" variable domain. Humanization can be essentially
performed following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent
CDRs or CDR sequences for the corresponding sequences of a human
antibody.

[0161] The choice of human variable domains, both light and heavy, to be
used in making the humanized antibodies is very important to reduce
antigenicity. According to the so-called "best-fit" method, the sequence
of the variable domain of a rodent antibody, is screened against the
entire library of known human variable-domain sequences. The human
sequence which is closest to that of the rodent is then accepted as the
human framework (FR) for the humanized antibody (Sims et al., J.
Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901
(1987)). Another method uses a particular framework derived from the
consensus sequence of all human antibodies of a particular subgroup of
light or heavy chains. The same framework can be used for several
different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

[0162] It is further important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties, for example, the ability to inhibit SGK1 activity and/or
function. To achieve this goal, humanized antibodies are prepared by a
process of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art. Computer programs
are available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of the
residues in the functioning of the candidate immunoglobulin sequence,
i.e., the analysis of residues that influence the ability of the
candidate immunoglobulin to bind its antigen. In this way, FR residues
can be selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased affinity for
the target antigen(s), is achieved. In general, the CDR residues are
directly and most substantially involved in influencing antigen binding.

[0163] Alternatively, it is possible to produce transgenic animals (e.g.,
mice) that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (JH) gene in
chimeric and germ-line mutant mice results in complete inhibition of
endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result in
the production of human antibodies upon antigen challenge. See, e.g.,
Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits
et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and Duchosal et al. Nature 355:258 (1992).

[0164] Alternatively, phage display technology (McCafferty et al., Nature
348:552-553 (1990)) can be used to produce human antibodies and antibody
fragments in vitro, from immunoglobulin variable (V) domain gene
repertoires from unimmunized donors. According to this technique,
antibody V domain genes are cloned in-frame into either a major or minor
coat protein gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the phage
particle. Because the filamentous particle contains a single-stranded DNA
copy of the phage genome, selections based on the functional properties
of the antibody also result in selection of the gene encoding the
antibody exhibiting those properties. Thus, the phage mimics some of the
properties of the B-cell. Phage display can be performed in a variety of
formats; for their review see, e.g., Johnson, Kevin S, and Chiswell,
David J., Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson et
al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library of V
genes derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to a
diverse array of antigens (including self-antigens) can be isolated
essentially following the techniques described by Marks et al., J. Mol.
Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).
See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

[0165] Human antibodies can also be generated by in vitro activated B
cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0166] Antibody Fragments.

[0167] In some embodiments of the aspects described herein, an antibody
specific for SGK1 can be treated or processed into an antibody fragment
thereof. Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,
Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and
Brennan et al., Science, 229:81 (1985)). However, these fragments can now
be produced directly by recombinant host cells. For example, the antibody
fragments can be isolated from the antibody phage libraries discussed
above. Alternatively, Fab'-SH fragments can be directly recovered from E.
coli and chemically coupled to form F(ab')2 fragments (Carter et
al., Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab')2 fragments can be isolated directly from recombinant host
cell culture. Other techniques for the production of antibody fragments
will be apparent to the skilled practitioner. In other embodiments, the
antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185.

Pharmaceutical Formulations of SGK1 Inhibitors and SGK1 Activators

[0168] Therapeutic formulations of the SGK1 inhibitors and SGK1 activators
described herein can be prepared, in some aspects, by mixing an SGK1
inhibitor, such as a small molecule SGK1 inhibitor of Formula (I) or
Formula (Ia), blocking or inhibitory SGK1 antibody or antigen-binding
fragment thereof, or nucleic acid-based SGK1 inhibitor, or an SGK1
activator described herein, having the desired degree of purity with one
or more pharmaceutically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),
in the form of lyophilized formulations or aqueous solutions. Such
therapeutic formulations of the SGK1 inhibitors and SGK1 activators
described herein include formulation into pharmaceutical compositions or
pharmaceutical formulations for parenteral administration, e.g.,
intravenous; mucosal, e.g., intranasal; enteral, e.g., oral; topical,
e.g., transdermal; ocular, or other mode of administration.

[0169] As used herein, the phrase "pharmaceutically acceptable" refers to
those compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in contact
with the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio. The phrase
"pharmaceutically acceptable carrier" as used herein means a
pharmaceutically acceptable material, composition or vehicle, such as a
liquid or solid filler, diluent, excipient, solvent, media, encapsulating
material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or
zinc stearate, or steric acid), or solvent encapsulating material,
involved in maintaining the activity, function of, solubility of, and/or
stability of a SGK1 modulator as described herein.

[0171] In some embodiments, a therapeutic formulation comprising an SGK1
inhibitor or an SGK1 activator comprises a pharmaceutically acceptable
salt, typically, e.g., sodium chloride, and preferably at about
physiological concentrations. Optionally, the formulations described
herein can contain a pharmaceutically acceptable preservative. In some
embodiments, the preservative concentration ranges from 0.1 to 2.0%,
typically v/v. Suitable preservatives include those known in the
pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben, and
propylparaben are examples of preservatives. Optionally, the formulations
described herein can include a pharmaceutically acceptable surfactant at
a concentration of 0.005 to 0.02%.

[0172] In some embodiments, an SGK1 modulator, such as an SGK1 inhibitor
or an SGK1 activator described herein, can be specially formulated for
administration of the compound to a subject in solid, liquid or gel form,
including those adapted for the following: (1) oral administration, for
example, drenches (aqueous or non-aqueous solutions or suspensions),
lozenges, dragees, capsules, pills, tablets (e.g., those targeted for
buccal, sublingual, and systemic absorption), boluses, powders, granules,
pastes for application to the tongue; (2) parenteral administration, for
example, by subcutaneous, intramuscular, intravenous or epidural
injection as, for example, a sterile solution or suspension, or
sustained-release formulation; (3) topical application, for example, as a
cream, ointment, or a controlled-release patch or spray applied to the
skin; (4) intravaginally or intrarectally, for example, as a pessary,
cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8)
transmucosally; or (9) nasally. Additionally, an SGK1 modulator, such as
an SGK1 inhibitor or an SGK1 activator described herein, can be implanted
into a patient or injected using a drug delivery system. See, for
example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236
(1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals"
(Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat.
No. 35 3,270,960. Examples of dosage forms include, but are not limited
to: tablets; caplets; capsules, such as hard gelatin capsules and soft
elastic gelatin capsules; cachets; troches; lozenges; dispersions;
suppositories; ointments; cataplasms (poultices); pastes; powders;
dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal
sprays or inhalers); gels; liquids such as suspensions (e.g., aqueous or
non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil
liquid emulsions), solutions, and elixirs; and sterile solids (e.g.,
crystalline or amorphous solids) that can be reconstituted to provide
liquid dosage forms.

[0173] In some embodiments, parenteral dosage forms of the SGK1
modulators, such as SGK1 inhibitors or SGK1 activators described herein,
can be administered to a subject in need of treatment, such as a subject
having an autoimmune disorder, or a subject having an infectious disease,
by various routes, including, but not limited to, subcutaneous,
intravenous (including bolus injection), intramuscular, and
intraarterial. Since administration of parenteral dosage forms typically
bypasses the patient's natural defenses against contaminants, parenteral
dosage forms are preferably sterile or capable of being sterilized prior
to administration to a patient. Examples of parenteral dosage forms
include, but are not limited to, solutions ready for injection, dry
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
controlled-release parenteral dosage forms, and emulsions.

[0174] Suitable vehicles that can be used to provide parenteral dosage
forms described herein are well known to those skilled in the art.
Examples include, without limitation: sterile water; water for injection
USP; saline solution; glucose solution; aqueous vehicles such as but not
limited to, sodium chloride injection, Ringer's injection, dextrose
Injection, dextrose and sodium chloride injection, and lactated Ringer's
injection; water-miscible vehicles such as, but not limited to, ethyl
alcohol, polyethylene glycol, and propylene glycol; and non-aqueous
vehicles such as, but not limited to, corn oil, cottonseed oil, peanut
oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0175] Due to their ease of administration, tablets and capsules represent
the most advantageous solid oral dosage unit forms, in which case solid
pharmaceutical excipients are used. If desired, tablets can be coated by
standard aqueous or nonaqueous techniques. These dosage forms can be
prepared by any of the methods of pharmacy. In general, pharmaceutical
compositions and dosage forms are prepared by uniformly and intimately
admixing the active ingredient(s) with liquid carriers, finely divided
solid carriers, or both, and then shaping the product into the desired
presentation if necessary.

[0176] Typical oral dosage forms of the compositions are prepared by
combining the pharmaceutically acceptable salt of an SGK1 modulator, such
as an SGK1 inhibitor or an SGK1 activator described herein, in an
intimate admixture with at least one excipient according to conventional
pharmaceutical compounding techniques. Excipients can take a wide variety
of forms depending on the form of the composition desired for
administration. For example, excipients suitable for use in oral liquid
or aerosol dosage forms include, but are not limited to, water, glycols,
oils, alcohols, flavoring agents, preservatives, and coloring agents.
Examples of excipients suitable for use in solid oral dosage forms (e.g.,
powders, tablets, capsules, and caplets) include, but are not limited to,
starches, sugars, microcrystalline cellulose, kaolin, diluents,
granulating agents, lubricants, binders, and disintegrating agents.

[0178] Examples of fillers suitable for use in the pharmaceutical
formulations described herein include, but are not limited to, talc,
calcium carbonate (e.g., granules or powder), microcrystalline cellulose,
powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,
starch, pre-gelatinized starch, and mixtures thereof. The binder or
filler in pharmaceutical compositions described herein is typically
present in from about 50 to about 99 weight percent of the pharmaceutical
composition.

[0179] Disintegrants are used in the oral pharmaceutical formulations
described herein to provide tablets that disintegrate when exposed to an
aqueous environment. A sufficient amount of disintegrant that is neither
too little nor too much to detrimentally alter the release of the active
ingredient(s) should be used to form solid oral dosage forms of the SGK1
modulators, such as the SGK1 inhibitors or the SGK1 activators described
herein. The amount of disintegrant used varies based upon the type of
formulation, and is readily discernible to those of ordinary skill in the
art. Disintegrants that can be used to form oral pharmaceutical
formulations include, but are not limited to, agar, alginic acid, calcium
carbonate, microcrystalline cellulose, croscarmellose sodium,
crospovidone, polacrilin potassium, sodium starch glycolate, potato or
tapioca starch, other starches, pre-gelatinized starch, clays, other
algins, other celluloses, gums, and mixtures thereof.

[0181] In other embodiments, lactose-free pharmaceutical formulations and
dosage forms are provided, wherein such compositions preferably contain
little, if any, lactose or other mono- or di-saccharides. As used herein,
the term "lactose-free" means that the amount of lactose present, if any,
is insufficient to substantially increase the degradation rate of an
active ingredient. Lactose-free compositions of the disclosure can
comprise excipients which are well known in the art and are listed in the
USP(XXI)/NF (XVI), which is incorporated herein by reference.

[0182] The oral formulations of the SGK1 modulators, such as the SGK1
inhibitors or the SGK1 activators described herein, further encompass, in
some embodiments, anhydrous pharmaceutical compositions and dosage forms
comprising these SGK1 inhibitors or SGK1 activators as active
ingredients, since water can facilitate the degradation of some
compounds. For example, the addition of water (e.g., 5%) is widely
accepted in the pharmaceutical arts as a means of simulating long-term
storage in order to determine characteristics such as shelf life or the
stability of formulations over time. See, e.g., Jens T. Carstensen, Drug
Stability: Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY,
N.Y.: 1995). Anhydrous pharmaceutical compositions and dosage forms
described herein can be prepared using anhydrous or low moisture
containing ingredients and low moisture or low humidity conditions.
Pharmaceutical compositions and dosage forms that comprise lactose and at
least one active ingredient that comprises a primary or secondary amine
are preferably anhydrous if substantial contact with moisture and/or
humidity during manufacturing, packaging, and/or storage is expected.
Anhydrous compositions are preferably packaged using materials known to
prevent exposure to water such that they can be included in suitable
formulary kits. Examples of suitable packaging include, but are not
limited to, hermetically sealed foils, plastics, unit dose containers
(e.g., vials) with or without desiccants, blister packs, and strip packs.

[0183] An SGK1 modulator, such as an SGK1 inhibitor or an SGK1 activator
described herein, can be administered directly to the airways in the form
of an aerosol or by nebulization. Accordingly, for use as aerosols, in
some embodiments, an SGK1 modulator may be packaged in a pressurized
aerosol container together with suitable propellants, for example,
hydrocarbon propellants like propane, butane, or isobutane with
conventional adjuvants. In other embodiments, the SGK1 inhibitor or the
SGK1 activator inhibitor can be administered in a non-pressurized form
such as in a nebulizer or atomizer.

[0184] The term "nebulization" is well known in the art to include
reducing liquid to a fine spray. Preferably, by such nebulization small
liquid droplets of uniform size are produced from a larger body of liquid
in a controlled manner. Nebulization can be achieved by any suitable
means, including by using many nebulizers known and marketed today. As is
well known, any suitable gas can be used to apply pressure during the
nebulization, with preferred gases being those which are chemically inert
to the SGK1 modulator, such as the SGK1 inhibitors or the SGK1 activators
described herein. Exemplary gases include, but are not limited to,
nitrogen, argon or helium.

[0185] In other embodiments, an SGK1 modulator, such as an SGK1 inhibitor
or an SGK1 activator described herein, can be administered directly to
the airways in the form of a dry powder. For use as a dry powder, an SGK1
inhibitor or an SGK1 activator can be administered by use of an inhaler.
Exemplary inhalers include metered dose inhalers and dry powdered
inhalers.

[0186] Suitable powder compositions include, by way of illustration,
powdered preparations of an SGK1 modulator, such as an SGK1 inhibitor or
an SGK1 activator described herein, thoroughly intermixed with lactose,
or other inert powders acceptable for, e.g., intrabronchial
administration. The powder compositions can be administered via an
aerosol dispenser or encased in a breakable capsule which may be inserted
by the subject into a device that punctures the capsule and blows the
powder out in a steady stream suitable for inhalation. The compositions
can include propellants, surfactants, and co-solvents and may be filled
into conventional aerosol containers that are closed by a suitable
metering valve.

[0188] Topical dosage forms of the SGK1 modulators, such as the SGK1
inhibitors or the SGK1 activators described herein, are also provided in
some embodiments, and include, but are not limited to, creams, lotions,
ointments, gels, shampoos, sprays, aerosols, solutions, emulsions, and
other forms known to one of skill in the art. See, e.g., Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990);
and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,
Philadelphia, Pa. (1985). For non-sprayable topical dosage forms, viscous
to semi-solid or solid forms comprising a carrier or one or more
excipients compatible with topical application and having a dynamic
viscosity preferably greater than water are typically employed. Suitable
formulations include, without limitation, solutions, suspensions,
emulsions, creams, ointments, powders, liniments, salves, and the like,
which are, if desired, sterilized or mixed with auxiliary agents (e.g.,
preservatives, stabilizers, wetting agents, buffers, or salts) for
influencing various properties, such as, for example, osmotic pressure.
Other suitable topical dosage forms include sprayable aerosol
preparations wherein the active ingredient, preferably in combination
with a solid or liquid inert carrier, is packaged in a mixture with a
pressurized volatile (e.g., a gaseous propellant, such as freon), or in a
squeeze bottle. Moisturizers or humectants can also be added to
pharmaceutical compositions and dosage forms if desired. Examples of such
additional ingredients are well known in the art. See, e.g., Remington's
Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa.
(1990). and Introduction to Pharmaceutical Dosage Forms, 4th Ed., Lea &
Febiger, Philadelphia, Pa. (1985). Dosage forms suitable for treating
mucosal tissues within the oral cavity can be formulated as mouthwashes,
as oral gels, or as buccal patches. Additional transdermal dosage forms
include "reservoir type" or "matrix type" patches, which can be applied
to the skin and worn for a specific period of time to permit the
penetration of a desired amount of active ingredient.

[0189] Examples of transdermal dosage forms and methods of administration
that can be used to administer an SGK1 modulator, such as an SGK1
inhibitor or an SGK1 activator described herein, include, but are not
limited to, those disclosed in U.S. Pat. Nos. 4,624,665; 4,655,767;
4,687,481; 4,797,284; 4,810,499; 4,834,978; 4,877,618; 4,880,633;
4,917,895; 4,927,687; 4,956,171; 5,035,894; 5,091,186; 5,163,899;
5,232,702; 5,234,690; 5,273,755; 5,273,756; 5,308,625; 5,356,632;
5,358,715; 5,372,579; 5,421,816; 5,466,465; 5,494,680; 5,505,958;
5,554,381; 5,560,922; 5,585,111; 5,656,285; 5,667,798; 5,698,217;
5,741,511; 5,747,783; 5,770,219; 5,814,599; 5,817,332; 5,833,647;
5,879,322; and 5,906,830, each of which are incorporated herein by
reference in their entirety.

[0190] Suitable excipients (e.g., carriers and diluents) and other
materials that can be used to provide transdermal and mucosal dosage
forms of the inhibitors described herein are well known to those skilled
in the pharmaceutical arts, and depend on the particular tissue or organ
to which a given pharmaceutical composition or dosage form will be
applied. In addition, depending on the specific tissue to be treated,
additional components may be used prior to, in conjunction with, or
subsequent to treatment with an SGK1 modulator, such as an SGK1 inhibitor
or an SGK1 activator described herein. For example, penetration enhancers
can be used to assist in delivering the active ingredients to or across
the tissue.

[0191] In some embodiments of the aspects described herein, the
pharmaceutical formulations comprising the SGK1 modulators, such as the
SGK1 inhibitors or the SGK1 activators described herein, can further
comprise more than one active compound as necessary for the particular
indication being treated, preferably those with complementary activities
that do not adversely affect each other. In other embodiments, the
formulation comprising the SGK1 modulator, such as an SGK1 inhibitor or
an SGK1 activator, can comprise a cytotoxic agent, cytokine, a cytokine
inhibitory agent, or a growth inhibitory agent. Such molecules are
suitably present in combination in amounts that are effective for the
purpose intended.

[0192] In some embodiments, the active ingredients of the formulations
comprising SGK1 modulators, such as the SGK1 inhibitors or the SGK1
activators described herein, can also be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example, liposomes,
albumin microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0193] In some embodiments, the SGK1 modulator, such as the SGK1
inhibitors or the SGK1 activators described herein, can be administered
to a subject by controlled- or delayed-release means. Ideally, the use of
an optimally designed controlled-release preparation in medical treatment
is characterized by a minimum of drug substance being employed to cure or
control the condition in a minimum amount of time. Advantages of
controlled-release formulations include: 1) extended activity of the
drug; 2) reduced dosage frequency; 3) increased patient compliance; 4)
usage of less total drug; 5) reduction in local or systemic side effects;
6) minimization of drug accumulation; 7) reduction in blood level
fluctuations; 8) improvement in efficacy of treatment; 9) reduction of
potentiation or loss of drug activity; and 10) improvement in speed of
control of diseases or conditions. (Kim, Cherng-ju, Controlled Release
Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)).
Controlled-release formulations can be used to control an SGK1
inhibitor's or activator's onset of action, duration of action, plasma
levels within the therapeutic window, and peak blood levels. In
particular, controlled- or extended-release dosage forms or formulations
can be used to ensure that the maximum effectiveness of the SGK1
modulators, such as the SGK1 inhibitors or the SGK1 activators described
herein, is achieved while minimizing potential adverse effects and safety
concerns, which can occur both from under-dosing a drug (i.e., going
below the minimum therapeutic levels) as well as exceeding the toxicity
level for the drug.

[0194] A variety of known controlled- or extended-release dosage forms,
formulations, and devices can be adapted for use with the SGK1
modulators, such as the SGK1 inhibitor or the SGK1 activators described
herein. Examples include, but are not limited to, those described in U.S.
Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719;
5674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;
5,354,556; 5,733,566; and 6,365,185 Bl; each of which is incorporated ins
entirety herein by reference. These dosage forms can be used to provide
slow or controlled-release of one or more active ingredients using, for
example, hydroxypropylmethyl cellulose, other polymer matrices, gels,
permeable membranes, osmotic systems (such as OROS® (Alza
Corporation, Mountain View, Calif. USA)), multilayer coatings,
microparticles, liposomes, or microspheres or a combination thereof to
provide the desired release profile in varying proportions. Additionally,
ion exchange materials can be used to prepare immobilized, adsorbed salt
forms of the disclosed compounds and thus effect controlled delivery of
the drug. Examples of specific anion exchangers include, but are not
limited to, Duolite® A568 and Duolite® AP143 (Rohm&Haas, Spring
House, Pa. USA).

[0195] In some embodiments, the SGK1 modulators, such as the SGK1
inhibitors or the SGK1 activator described herein, for use in the various
therapeutic formulations and compositions, and methods thereof, are
administered to a subject by sustained release or in pulses. Pulse
therapy is not a form of discontinuous administration of the same amount
of a composition over time, but comprises administration of the same dose
of the composition at a reduced frequency or administration of reduced
doses. Sustained release or pulse administrations are particularly
preferred in chronic conditions, such as autoimmune disorders or chronic
inflammatory conditions, as each pulse dose can be reduced and the total
amount of a compound of an, e.g., SGK1 inhibitor, such as a small
molecule of Formula (I) or Formula (Ia), administered over the course of
treatment to the patient is minimized.

[0196] The interval between pulses, when necessary, can be determined by
one of ordinary skill in the art. Often, the interval between pulses can
be calculated by administering another dose of the composition when the
composition or the active component of the composition is no longer
detectable in the subject prior to delivery of the next pulse. Intervals
can also be calculated from the in vivo half-life of the composition.
Intervals may be calculated as greater than the in vivo half-life, or 2,
3, 4, 5 and even 10 times greater the composition half-life. Various
methods and apparatus for pulsing compositions by infusion or other forms
of delivery to the patient are disclosed in U.S. Pat. Nos. 4,747,825;
4,723,958; 4,948,592; 4,965,251 and 5,403,590.

[0197] In some embodiments, sustained-release preparations comprising the
SGK1 modulator, such as an SGK1 inhibitor or an SGK1 activator described
herein, can be prepared. Suitable examples of sustained-release
preparations include semipermeable matrices of solid hydrophobic polymers
containing the inhibitor, in which matrices are in the form of shaped
articles, e.g., films, or microcapsule. Examples of sustained-release
matrices include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides
(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y
ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable
lactic acid-glycolic acid copolymers such as the LUPRON DEPOT®
(injectable microspheres composed of lactic acid-glycolic acid copolymer
and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0198] The formulations comprising the SGK1 modulators, such as the SGK1
inhibitors or the SGK1 activators described herein, to be used for in
vivo administration are preferably sterile. This is readily accomplished
by filtration through, for example, sterile filtration membranes, and
other methods known to one of skill in the art.

Modulating TH17-Mediated Immune Responses by SGK1 Modulation

[0199] Certain aspects of the methods described herein are based, in part,
on the discovery by the inventors that TH17 differentiation and
maintenance of TH17 cells is impaired or inhibited in the absence of SGK1
expression and/or activity. Thus, in some aspects described herein are
SGK1 inhibitors for inhibiting TH17 differentiation and activity, and
inhibiting TH17-mediated immune responses. In other aspects, described
herein are SGK1 activators for promoting or increasing TH17
differentiation and activity, and promoting and increasing TH17-mediated
immune responses. Accordingly, the methods using the SGK1 inhibitors and
SGK1 activators described herein are useful in the treatment of subjects
having diseases or disorders mediated or modulated by TH17 expression and
or activity, such as autoimmunity, chronic inflammatory disorders,
infectious diseases, cancer, allergic conditions, and the like.

[0200] A "TH17-mediated immune response" refers to an immune response that
is associated with the induction of, differentiation of, expansion of,
proliferation of, functional activity of, or a combination thereof, one
or more TH17 cells. At a minimum, as used herein, a "TH17 cell" refers to
a CD4+ T cell that expresses and/or produces IL-17A, also known herein as
"IL-17." In some embodiments, a TH17 cell is further characterized by
expression of one or more cytokines selected from the following: IL-17F,
IL-22, IL-26, IL-21, and TNF-α. In some embodiments, a TH17 cell is
further characterized by cell-surface expression of the chemokine
receptor CCR6. In some embodiments, a TH17 cell is further characterized
by cell-surface expression of the chemokine receptors CCR6 and CCR4. In
some embodiments, a TH17 cell is further characterized by cell-surface
expression of the chemokine receptor CCR6 and IL23R. In some embodiments,
a TH17 cell is further characterized by cell-surface expression of the
C-type lectin CD161. In some embodiments, a TH17 cell can be further
characterized by expression or activity of one or more of the following
factors: RORγt, RORα, STAT3, IRF4, the AhR (aryl hydrocarbon
receptor), and BATf. In some embodiments, a TH17 cell can be further
characterized as a cell expressing or producing IL-17, but not expressing
or producing certain cytokines, such as IL-4, IL-5, and IFN-γ. In
some embodiments, a TH17 cell can be further characterized as a cell
expressing or producing IL-17, but not expressing or producing certain
transcription factors such as T-bet, GATA-3, FOXP3, STAT1, STAT4, and
STAT5.

[0201] TH17 cells as described herein can be generated or propagated under
a variety of conditions. In some embodiments, a TH17 cell can be
generated or derived from a naive CD4 T cell in the presence of
TGF-β and IL-6. In some embodiments, a TH17 cell can be generated or
derived from a naive CD4 T cell in the presence of TGF-β and IL-21.
In other embodiments, a TH17 cell or a population of TH17 cells is
generated or derived from expansion of a population of TH17 cells in the
presence of IL-23. In other embodiments, a population of TH17 cells can
be maintained in the presence of IL-23.

[0202] Accordingly, in some aspects and embodiments described herein,
inhibiting a TH17-mediated immune response refers to inhibition of IL-17
expression and/or production by a TH17 cell. In some embodiments,
inhibiting a TH17-mediated immune response refers to inhibition of the
expression and/or production of IL-17 by a TH17 cell and inhibition of
the expression and/or production of one or more of the following
cytokines: IL-17F, IL-22, IL-26, IL-21, and TNF-α by a TH17 cell.
In such embodiments, inhibition of cytokine production can be assayed
using any of a number of methods known to one of skill in the art. For
example, biological samples, such as a peripheral blood sample, a serum
sample, or a cerebrospinal fluid sample, can be obtained from a subject
before and after treatment or administration of an SGK1 modulator as
described herein. A statistically significant decrease in the number of
cytokine producing cells, as measured, for example, by flow cytometric
analysis of TH17 producing cells, is indicative of an inhibition of a
TH17 mediated immune response. Cytokine production (and a decrease or
inhibition thereof) can also be ascertained in a biological sample, such
as serum, using ELISA, bead-based cytokine assays, or any such assay as
known to one of skill in the art for measuring cytokine levels.

[0203] In some embodiments, inhibiting a TH17-mediated immune response
refers to inhibition of proliferation of or expansion of a TH17 cell. In
some embodiments, inhibiting a TH17-mediated immune response refers to
inhibiting the differentiation of a CD4+ T cell or a population of
CD4+ T cells into a TH17 cell or population of TH17 cells. In such
embodiments, changes in proliferation or expansion of a TH17 cell
population, or changes in differentiation of a population of a CD4+
T cell can be determined using any of a number of assays, including
measurement of the number of IL-17 producing cells in biological samples
obtained from a subject before and after contacting with, treatment with,
or administration of an SGK1 modulator, as described herein. Thus, a
reduction in the number of TH17 cells in a contacted, treated, or
administered sample relative to a sample prior to such contacting,
treating, or administering is indicative of inhibition of the TH17
mediated immune response.

[0205] Accordingly, in one aspect, described herein are methods for
inhibiting differentiation of a precursor CD4+ T cell or a CD4+
T cell population into a TH17 cell or TH17 cell population. Such methods
comprise contacting a CD4+ T cell or CD4+ T cell population
with an inhibitor or antagonist of SGK1 in an amount sufficient to
inhibit TH17 cell differentiation. In some embodiments of these methods,
the methods of inhibiting TH17 differentiation can further comprise
contacting a CD4+ T cell or CD4+ T cell population with an
inhibitor or antagonist of one or more of the following: TGF-β,
IL-6, IL-21, IL-23, RORγt, RORα, STAT3, IRF4, the AhR (aryl
hydrocarbon receptor), and BATf.

[0206] In another aspect, methods of inhibiting a TH17-mediated immune
response using an SGK1 inhibitor as described herein are provided. In
some embodiments of these methods, inhibiting a TH17-mediated immune
response refers to inhibition of IL-17 expression and/or production by a
TH17 cell. In some embodiments, inhibiting a TH17-mediated immune
response further comprises inhibition of expression and/or production of
one or more of the following cytokines: IL-17F, IL-22, IL-26, IL-21, and
TNF-α. In some embodiments of these methods, inhibiting a
TH17-mediated immune response refers to inhibition of proliferation of or
expansion of a TH17 cell. In some embodiments, inhibiting a TH17-mediated
immune response refers to inhibition of trafficking of a TH17 cell. Such
methods comprise contacting a CD4+ T cell or CD4+ T cell
population with an inhibitor or antagonist of SGK1 in an amount
sufficient to inhibit the TH17-mediated immune response. In some
embodiments of these methods, the methods of inhibiting a TH17-mediated
immune response can further comprise contacting a CD4+ T cell or
CD4+ T cell population with an inhibitor or antagonist of one or
more of the following: TGF-β, IL-6, IL-21, IL-23, RORγt,
RORα, STAT3, IRF4, the AhR (aryl hydrocarbon receptor), and BATf.
In some embodiments of these aspects, the contacting step can be carried
out ex vivo, in vitro, or in vivo. For example, in one embodiment, the
contacting step is performed using human cells, or performed in a human
patient.

[0207] By "inhibiting" a TH17-mediated immune response is meant that the
production or expression of IL-17 by a TH17 cell or TH17 cell population,
the rate of proliferation and/or expansion of a TH17 cell or TH17 cell
population, the number of cells differentiating into a TH17 cell, the
number or quantity of TH17 cells trafficking to a target tissue or site,
or any combination thereof, is at least 10% less, at least 15% less, at
least 20% less, at least 25% less, at least 30% less, at least 35% less
at least 40% less, at least 45% less, at least 50% less, at least 55%
less, at least 60% less, at least 65% less, at least 70% less, at least
75% less, at least 80% less, at least 85% less, at least 90% less, at
least 95% less, or completely absent or undetectable in comparison to a
reference or control level or sample in the absence of the SGK1
inhibitor.

[0208] Accordingly, in one aspect, described herein are methods for
inhibiting TH17-mediated immune responses in a subject in need thereof.
Such methods comprise administering to a subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising an inhibitor of SGK1 expression and/or SGK1 activity for
inhibiting TH17-mediated immune responses. In some embodiments of these
aspects, a subject in need of inhibition of a TH17-mediated immune
response has or is at risk for a TH17-mediated disorder. As used herein,
a "TH17-mediated disorder" or "TH17-mediated disease" refer to a disease
or disorder that is caused by (in part or fully), associated with, or
exacerbated by, the development of a TH17-mediated immune response. In
such TH17-mediated disorders, inhibition and/or reduction in the
TH17-mediated immune response provides a beneficial effect to the subject
being treated, i.e., ameliorates, cures, suppresses, delays, prevents the
onset of, prevents the recurrence or relapse of one or more of the
symptoms associated with the disease or disorder.

[0209] The terms "subject" and "individual" are used interchangeably
herein, and refer to an animal, for example a human, recipient of the
SGK1 inhibitors, such as the small molecules of Formula (I) or Formula
(Ia), RNA-based SGK1 inhibitors, or blocking anti-SGK1 antibodies or
antigen-binding fragments thereof, or, in other aspects, SGK1 activators
described herein. For treatment of those disease states which are
specific for a specific animal such as a human subject, the term
"subject" refers to that specific animal. The terms `non-human animals`
and `non-human mammals` are used interchangeably herein, and include
mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and
non-human primates. The term "subject" also encompasses any vertebrate
including but not limited to mammals, reptiles, amphibians and fish. In
some embodiments of the aspects described herein, a subject refers to a
human subject having an autoimmune disease. In some embodiments of the
aspects described herein, a subject refers to a human subject having a
chronic inflammatory disease. In some embodiments of the aspects
described herein, a subject refers to a human subject having an
infectious disease.

[0210] In some embodiments of these aspects, the TH17-mediated disease or
disorder is an autoimmune disorder. In some embodiments, the methods
further comprise selecting or identifying a subject having a
TH17-mediated autoimmune disease or disorder.

[0211] An "autoimmune disorder" or an "autoimmune disease" as the terms
are used herein refer to those disorders or diseases that are the result
of inappropriate activation of immune cells that are reactive against
self tissue, and which are characterized by the production of cytokines,
such as IL-17, and autoantibodies involved in the pathology of the
diseases. Preventing the activation or effector function, such as IL-17
production, of autoreactive immune cells can reduce or eliminate disease
symptoms. Accordingly, in some embodiments, an autoreactive immune cell
is a an autoreactive TH17 cell. Non-limiting examples of autoimmune
diseases include multiple sclerosis, rheumatoid arthritis, Crohn's
disease, systemic lupus erythematosus (SLE), autoimmune
encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,
Goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris), Grave's
disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic
purpura, scleroderma (e.g., with anti-collagen antibodies), mixed
connective tissue disease, polymyositis, pernicious anemia, idiopathic
Addison's disease, autoimmune-associated infertility, glomerulonephritis
(e.g., crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus (type 1 diabetes mellitus; insulin-dependent
diabetes mellitus). Most autoimmune diseases are also encompassed within
the term "chronic inflammatory diseases." Such diseases or disorders are
processes associated with long-term (>6 months) activation of
inflammatory immune cells, such as TH17 cells. The chronic inflammation
leads to damage of patient organs or tissues. In addition to autoimmune
disorders, many diseases are considered to be chronic inflammatory
disorders, but are not currently known to have an autoimmune basis.
Examples include atherosclerosis, congestive heart failure, polyarteritis
nodosa, Whipple's Disease, and primary sclerosing cholangitis.

[0212] In another aspect, described herein are methods for promoting
TH17-mediated immune responses in a subject in need thereof. Such methods
comprise administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising an activator
of SGK1 expression and/or SGK1 activity for increasing or promoting
TH17-mediated immune responses. In such aspects, activating, increasing,
or promoting the TH17-mediated immune response provides a beneficial
effect to the subject being treated. For example, increasing the
TH17-mediated response during an infectious disease or disorder in which
expression or production of IL-17 decreases the infectious load or
inhibits replication of the infectious agent are non-limiting examples of
beneficial effects mediated by a TH17 response.

[0213] In some embodiments of these aspects, the TH17-mediated immune
response refers to a TH17-mediated infectious immune response. Infectious
immune responses in which increased or enhanced TH17 responses are useful
include, but are not limited to, responses to extracellular infectious
pathogens such as Klebsiella pneumoniae, Staphylococcus aureus, and
Candida albicans. In some embodiments, the TH17-mediated immune response
refers to a TH17-mediated immune response at an epithelial surface. In
some embodiments of these aspects, a TH17-mediated immune response refers
to a TH17-mediated mucosal immune response.

[0214] In some embodiments, the subject being administered the activator
of SGK1 expression and/or SGK1 activity for increasing or promoting
TH17-mediated immune responses has a persistent infection with a
bacterium, virus, fungus, or parasite. "Persistent infections" refer to
those infections that, in contrast to acute infections, are not
effectively cleared by the induction of a host immune response. During
such persistent infections, the infectious agent and the immune response
reach equilibrium such that the infected subject remains infectious over
a long period of time without necessarily expressing symptoms. Persistent
infections often involve stages of both silent and productive infection
without rapidly killing or even producing excessive damage of the host
cells. Persistent infections include for example, latent, chronic and
slow infections. Persistent infection occurs with viruses including, but
not limited to, human T-Cell leukemia viruses, Epstein-Barr virus,
cytomegalovirus, herpesviruses, varicella-zoster virus, measles,
papovaviruses, prions, hepatitis viruses, adenoviruses, parvoviruses and
papillomaviruses.

[0215] The mechanisms by which persistent infections are maintained can
involve modulation of virus and cellular gene expression and modification
of the host immune response. Reactivation of a latent infection can be
triggered by various stimuli, including changes in cell physiology,
superinfection by another virus, and physical stress or trauma. Host
immunosuppression is often associated with reactivation of a number of
persistent virus infections.

[0217] Additional examples of fungal infections that can be treated using
the SGK1 activators described herein include but are not limited to:
aspergillosis; thrush (caused by Candida albicans); cryptococcosis
(caused by Cryptococcus); and histoplasmosis. Thus, examples of
infectious fungi include, but are not limited to, Cryptococcus
neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces
dermatitidis, Chlamydia trachomatis, Candida albicans. The compositions
and methods described herein are contemplated for use in treating
infections with these fungal agents.

[0220] In other embodiments of the methods described herein, the subject
having a TH17-mediated disorder has a cancer or tumor. A subject that has
a cancer or a tumor is a subject having objectively measurable cancer
cells present in the subject's body. In some such embodiments of the
aspects described herein, the cancer is a solid tumor. Accordingly,
provided herein are methods to treat a subject having a cancer or tumor
comprising administering a therapeutically effective amount of an SGK1
modulator for modulating TH17-mediated immune responses in the subject
having a cancer or a tumor. Depending on the nature of the cancer being
treated, in some embodiments, the SGK1 modulator is an SGK1 inhibitor. In
some embodiments, the SGK1 modulator is an SGK1 activator.

[0221] A "cancer" or "tumor" as used herein refers to an uncontrolled
growth of cells which interferes with the normal functioning of the
bodily organs and systems. A subject that has a cancer or a tumor is a
subject having objectively measurable cancer cells present in the
subject's body. Included in this definition are benign and malignant
cancers, as well as dormant tumors or micrometastatses. Cancers which
migrate from their original location and seed vital organs can eventually
lead to the death of the subject through the functional deterioration of
the affected organs. Hemopoietic cancers, such as leukemia, are able to
out-compete the normal hemopoietic compartments in a subject, thereby
leading to hemopoietic failure (in the form of anemia, thrombocytopenia
and neutropenia) ultimately causing death.

[0222] By "metastasis" is meant the spread of cancer from its primary site
to other places in the body. Cancer cells can break away from a primary
tumor, penetrate into lymphatic and blood vessels, circulate through the
bloodstream, and grow in a distant focus (metastasize) in normal tissues
elsewhere in the body. Metastasis can be local or distant. Metastasis is
a sequential process, contingent on tumor cells breaking off from the
primary tumor, traveling through the bloodstream, and stopping at a
distant site. At the new site, the cells establish a blood supply and can
grow to form a life-threatening mass. Both stimulatory and inhibitory
molecular pathways within the tumor cell regulate this behavior, and
interactions between the tumor cell and host cells in the distant site
are also significant.

[0223] Metastases are most often detected through the sole or combined use
of magnetic resonance imaging (MRI) scans, computed tomography (CT)
scans, blood and platelet counts, liver function studies, chest X-rays
and bone scans in addition to the monitoring of specific symptoms.

[0225] In some embodiments of these aspects, the methods of treating
cancer or a cancerous condition using the SGK1 modulators, such as the
SGK1 inhibitors or SGK1 activators described herein, further comprise the
step of selecting or identifying a subject having cancer. In such
embodiments, a subject is identified as having cancer by objective
determination of the presence of cancer cells or a tumor in the subject's
body by one of skill in the art. Such objective determinations can be
performed through the sole or combined use of tissue biopsies, blood and
platelet cell counts, urine analyses, magnetic resonance imaging (MRI)
scans, computed tomography (CT) scans, liver function studies, chest
X-rays and bone scans in addition to the monitoring of specific symptoms
associated with a cancer.

[0226] In some embodiments described herein, the methods further comprise
administering a tumor or cancer antigen to a subject being administered
the SGK1 modulator. A number of tumor antigens have been identified that
are associated with specific cancers. As used herein, the terms "tumor
antigen" and "cancer antigen" are used interchangeably to refer to
antigens which are differentially expressed by cancer cells and can
thereby be exploited in order to target cancer cells. Cancer antigens are
antigens which can potentially stimulate apparently tumor-specific immune
responses. Some of these antigens are encoded, although not necessarily
expressed, by normal cells. These antigens can be characterized as those
which are normally silent (i.e., not expressed) in normal cells, those
that are expressed only at certain stages of differentiation and those
that are temporally expressed such as embryonic and fetal antigens. Other
cancer antigens are encoded by mutant cellular genes, such as oncogenes
(e.g., activated ras oncogene), suppressor genes (e.g., mutant p53),
fusion proteins resulting from internal deletions or chromosomal
translocations. Still other cancer antigens can be encoded by viral genes
such as those carried on RNA and DNA tumor viruses. Many tumor antigens
have been defined in terms of multiple solid tumors: MAGE 1, 2, & 3,
defined by immunity; MART-1/Melan-A, gp100, carcinoembryonic antigen
(CEA), HER-2, mucins (i.e., MUC-1), prostate-specific antigen (PSA), and
prostatic acid phosphatase (PAP). In addition, viral proteins such as
hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV) have
been shown to be important in the development of hepatocellular
carcinoma, lymphoma, and cervical cancer, respectively. However, due to
the immunosuppression of patients diagnosed with cancer, the immune
systems of these patients often fail to respond to the tumor antigens.

[0228] In other embodiments of the methods described herein, the subject
in need of modulation of a TH17-mediated immune response has any of the
following conditions or disorders: atopic conditions, such as asthma and
allergy, including allergic rhinitis, gastrointestinal allergies,
including food allergies, eosinophilia, conjunctivitis, In some such
embodiments, the SGK1 modulator is an SGK1 inhibitor. In other
embodiments, the SGK1 modulator is an SGK1 activator.

Administration, Dosages, and Durations

[0229] An SGK1 modulator, such as the SGK1 inhibitors, e.g., small
molecule SGK1 inhibitors of Formula (I) or Formula (Ia) or other small
molecule SGK1 inhibitors, RNA-based SGK1 inhibitors, and blocking
anti-SGK1 antibodies or antigen-binding fragments described herein, and
the SGK1 activators, such as activating anti-SGK1 antibodies and
antigen-binding fragments thereof, can be formulated, dosed, and
administered in a fashion consistent with good medical practice for use
in the treatment of the TH17-mediated disorders described herein, such as
autoimmune disorders. Factors for consideration in this context include
the particular disorder or type of disorder being treated, the particular
subject being treated, the clinical condition of the individual subject,
the cause of the disorder, the site of delivery of the agent, the method
of administration, the scheduling of administration, and other factors
known to medical practitioners.

[0230] Accordingly, the "therapeutically effective amount" of an SGK1
modulator, such as the SGK1 inhibitors, e.g., small molecule SGK1
inhibitors of Formula (I) or Formula (Ia), and the SGK1 activators
described herein, to be administered is governed by such considerations,
and, as used herein, refers to the minimum amount necessary to prevent,
ameliorate, or treat, or stabilize, the TH17-mediated disorder. In some
embodiments, an SGK1 modulator, is optionally formulated with one or more
agents currently used to prevent or treat the disorder being treated. The
effective amount of such other agents depends on the amount of the SGK1
modulator present in the formulation, the type of disorder or treatment,
and other factors discussed herein, and as understood by one of skill in
the art. These are generally used in the same dosages and with
administration routes as used herein above or from about 1 to 99% of the
heretofore employed dosages.

[0231] An effective amount as used herein also includes an amount
sufficient to delay the development of a symptom of the TH17-mediated
disorder, alter the course of the TH17-mediated disorder (for example but
not limited to, inhibit or delay time until relapse in
relapsing-remitting multiple sclerosis), or reverse a symptom of the
autoimmune disease or disorder. Thus, it is not possible to specify an
exact "effective amount". However, for any given case, an appropriate
"effective amount" can be determined by one of ordinary skill in the art
using only routine experimentation. If a certain amount of an SGK1
modulator as described herein statistically significantly alters an
indicium of a TH17 response, e.g., decreases the number of TH17 cells,
reduces the production of IL-17, reduces the proliferation of TH17 cells,
and/or reduces trafficking of TH17 cells, as defined herein, it is
evidence that said amount is therapeutically effective.

[0232] Accordingly, as used herein, the terms "treat," "treatment,"
"treating," or "amelioration" refer to therapeutic treatments, wherein
the object is to reverse, alleviate, ameliorate, inhibit, slow down or
stop the progression or severity of a condition associated with, a
disease or disorder. The term "treating" includes reducing or alleviating
at least one adverse effect or symptom of a condition, disease or
disorder associated with a chronic immune condition, such as, but not
limited to, an autoimmune disorder, a chronic inflammatory disorder, an
infection, or a cancer. Treatment is generally "effective" if one or more
symptoms, clinical markers, or indicia of disease are reduced to a
clinically significant degree. Alternatively, treatment is "effective" if
the progression of a disease is reduced or halted. That is, "treatment"
includes not just the improvement of symptoms or markers, but also a
cessation or at least slowing of progress or worsening of symptoms that
would be expected in the absence of treatment. Beneficial or desired
clinical results include, but are not limited to, alleviation of one or
more symptom(s), diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission (whether
partial or total), whether detectable or undetectable. The term
"treatment" of a disease also includes providing relief from the symptoms
or side-effects of the disease (including palliative treatment).

[0233] For example, in some embodiments, the methods described herein
comprise administering an effective amount of the SGK1 inhibitors
described herein to a subject in order to alleviate one or more symptoms
of an autoimmune disorder. As used herein, "alleviating a symptom of an
autoimmune disorder" refers to ameliorating any condition or symptom
associated with the autoimmune disorder. Alternatively, alleviating a
symptom of an autoimmune disorder can involve reducing the number of
autoimmune cells in the subject relative to the number of autoimmune
cells in an untreated control. In some embodiments, the autoimmune cells
comprise TH17 cells. As compared with an equivalent untreated control,
such reduction or degree of prevention is at least 5%, 10%, 20%, 40%,
50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.
Desirably, the autoimmune disorder is completely abrogated, as detected
by any standard method known in the art, in which case the autoimmune
disorder is considered to have been cured. A patient who is being treated
for an autoimmune disorder is one who a medical practitioner has
diagnosed as having such a condition. Diagnosis can be by any suitable
means. Diagnosis and monitoring can involve, for example, detecting the
level of autoimmune cells or autoantibodies in a biological sample (for
example, a tissue biopsy, blood or serum test, or urine test), detecting
the level of a surrogate marker of the autoimmune disorder in a
biological sample, detecting symptoms associated with the autoimmune
disorder, or detecting immune cells involved in the immune response
typical of the autoimmune disorder (for example, detection of
self-antigen-specific T cells that secrete inflammatory cytokines, such
as IL17).

[0234] In other embodiments, the methods described herein comprise
administering an effective amount of the SGK1 inhibitors or SGK1
activators described herein to a subject in order to alleviate one or
more symptoms of a cancer or tumor in a subject in need thereof. As used
herein, "alleviating a symptom of a cancer" refers to ameliorating any
condition or symptom associated with the cancer. In preferred
embodiments, an SGK1 modulator described herein can produce marked
anti-cancer effects in a human subject without causing significant
toxicities or adverse effects. The efficacy of the SGK1 treatments
described herein can be measured by various parameters commonly used in
evaluating cancer treatments, including but not limited to, tumor
regression, tumor weight or size shrinkage, reduction in rate of tumor
growth, the presence or the size of a dormant tumor, the presence or size
of metastases or micrometastases, degree of tumor or cancer invasiveness,
size or number of the blood vessels, time to progression, duration of
survival, progression free survival, overall response rate, duration of
response, and quality of life.

[0235] Effective amounts, toxicity, and therapeutic efficacy of the SGK1
modulators, such as the SGK1 inhibitors, e.g., small molecule SGK1
inhibitors of Formula (I) or Formula (Ia), and the SGK1 activators
described herein, can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining the
LD50 (the dose lethal to 50% of the population) and the ED50
(the dose therapeutically effective in 50% of the population). The dosage
can vary depending upon the dosage form employed and the route of
administration utilized. The dose ratio between toxic and therapeutic
effects is the therapeutic index and can be expressed as the ratio
LD50/ED50. Compositions and methods that exhibit large
therapeutic indices are preferred. A therapeutically effective dose can
be estimated initially from cell culture assays. Also, a dose can be
formulated in animal models to achieve a circulating plasma concentration
range that includes the IC50 (i.e., the concentration of the SGK1
inhibitor or SGK1 activator), which achieves a half-maximal inhibition of
symptoms) as determined in cell culture, or in an appropriate animal
model. Levels in plasma can be measured, for example, by high performance
liquid chromatography. The effects of any particular dosage can be
monitored by a suitable bioassay. The dosage can be determined by a
physician and adjusted, as necessary, to suit observed effects of the
treatment.

[0236] Depending on the type and severity of the disease, about 1 μg/kg
to 100 mg/kg (e.g., 0.1-20 mg/kg) of e.g., a small molecule SGK1
inhibitor of Formula (I) or Formula (Ia) described herein, is an initial
candidate dosage range for administration to the subject, whether, for
example, by one or more separate administrations, or by continuous
infusion. A typical daily dosage might range from about 1 μg/kg to
about 100 mg/kg or more, depending on the factors mentioned above. For
repeated administrations over several days or longer, depending on the
condition, the treatment is sustained until the cancer is treated, as
measured by the methods described above or known in the art. However,
other dosage regimens may be useful. The progress of the therapeutic
methods described herein is easily monitored by conventional techniques
and assays, such as those described herein, or known to one of skill in
the art. In other embodiments, such dosing regimen is used in combination
with a chemotherapy regimen as the first line therapy for treating
locally recurrent or metastatic breast cancer.

[0237] The duration of the therapeutic methods described herein can
continue for as long as medically indicated or until a desired
therapeutic effect (e.g., those described herein) is achieved. In certain
embodiments, administration of an SGK1 modulator, i.e., "SGK1 inhibitor
therapy" or "SGK1 activator therapy" is continued for at least 1 month,
at least 2 months, at least 4 months, at least 6 months, at least 8
months, at least 10 months, at least 1 year, at least 2 years, at least 3
years, at least 4 years, at least 5 years, at least 10 years, at least 20
years, or for at least a period of years up to the lifetime of the
subject.

[0238] The SGK1 modulators described herein, such as the SGK1 inhibitors,
e.g., small molecule SGK1 inhibitors of Formula (I) or Formula (Ia),
other small molecule SGK1 inhibitors, RNA-based SGK1 inhibitors, and
blocking anti-SGK1 antibodies or antigen-binding fragments thereof, and
the SGK1 activators, can be administered to a subject, e.g., a human
subject, in accordance with known methods, such as intravenous
administration as a bolus or by continuous infusion over a period of
time, by intramuscular, intraperitoneal, intracerobrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical,
or inhalation routes. Local administration can be used if, for example,
extensive side effects or toxicity is associated with the SGK1 inhibitor
or the SGK1 activator. An ex vivo strategy can also be used for
therapeutic applications.

[0239] Exemplary modes of administration of the SGK1 modulators described
herein, such as the SGK1 inhibitors, e.g., small molecule SGK1 inhibitors
of Formula (I) or Formula (Ia), other small molecule SGK1 inhibitors,
RNA-based SGK1 inhibitors, and blocking anti-SGK1 antibodies or
antigen-binding fragments thereof, and the SGK1 activators, include, but
are not limited to, injection, infusion, inhalation (e.g., intranasal or
intratracheal), ingestion, rectal, and topical (including buccal and
sublingual) administration. The phrases "parenteral administration" and
"administered parenterally" as used herein, refer to modes of
administration other than enteral and topical administration, usually by
injection. As used herein, "injection" includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal, intraventricular,
intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,
subarachnoid, intraspinal, intracerebro spinal, and intrasternal
injection and infusion. The phrases "systemic administration,"
"administered systemically", "peripheral administration" and
"administered peripherally" as used herein refer to the administration of
an SGK1 modulator, such as the SGK1 inhibitors, e.g., small molecule SGK1
inhibitors of Formula (I) or Formula (Ia), and the SGK1 activators
described herein, other than directly into a target site, tissue, or
organ, such as the lung, such that it enters the subject's circulatory
system and, thus, is subject to metabolism and other like processes.

[0240] In some embodiments, the SGK1 modulators, such as the SGK1
inhibitors, e.g., small molecule SGK1 inhibitors of Formula (I) or
Formula (Ia), and the SGK1 activators described herein, are administered
by intravenous infusion or injection. In some embodiments, where local
treatment is desired, for example, at or near a site of a TH17-mediated
immune response, such as in a joint of a patient having rheumatoid
arthritis, the SGK1 modulators, such as the SGK1 inhibitors, e.g., small
molecule SGK1 inhibitors of Formula (I) or Formula (Ia), and the SGK1
activators can be administered by intralesional administration.
Additionally, in some embodiments, the SGK1 inhibitors or SGK1 activators
described herein can be administered by pulse infusion, particularly with
declining doses of the inhibitors or non-constitutive agonists.
Preferably the dosing is given by injections, most preferably intravenous
or subcutaneous injections, depending in part on whether the
administration is brief or chronic.

[0241] The present invention may also be defined in any of the following
numbered paragraphs: [0242] 1. A method of inhibiting differentiation
of a CD4+ T cell or a CD4+ T cell population into a TH17 cell
or TH17 cell population, the method comprising contacting a CD4+ T
cell or CD4+ T cell population with a serum and
glucocorticoid-regulated kinase 1 (SGK1) inhibitor in an amount
sufficient to inhibit TH17 cell differentiation. [0243] 2. The method of
paragraph 1, further comprising contacting the CD4+ T cell or
CD4+ T cell population with an inhibitor or antagonist of one or
more of the following molecules: TGF-β, IL-6, IL-21, IL-23,
RORγt, RORα, STAT3, IRF4, AhR (aryl hydrocarbon receptor),
and BATf. [0244] 3. A method of inhibiting a TH17 cell-mediated immune
response in a subject in need thereof, the method comprising
administering to a subject in need thereof a therapeutically effective
amount of a serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitor
to inhibit a TH17 cell-mediated immune response. [0245] 4. The method of
paragraph 3, wherein the TH17 cell-mediated response being inhibited
comprises expression or production of IL-17 by a TH17 cell. [0246] 5. The
method of any of paragraphs 3-4, wherein the TH17 cell-mediated response
being inhibited comprises expression or production of one or more of
IL-17F, IL-22, IL-26, IL-21, and TNF-α. [0247] 6. The method of any
of paragraphs 3-5, wherein the TH17 cell-mediated response being
inhibited comprises inhibition of proliferation of or expansion of a TH17
cell. [0248] 7. The method of any of paragraphs 3-6, wherein the TH17
cell-mediated response being inhibited comprises trafficking of a TH17
cell. [0249] 8. The method of any of paragraphs 3-7, wherein the subject
in need of inhibition of a TH17-mediated immune response has a
TH17-mediated disorder. [0250] 9. The method of paragraph 8, wherein the
TH17-mediated disorder is an autoimmune disease or a chronic inflammatory
disease. [0251] 10. The method of paragraph 9, wherein the autoimmune
disease is multiple sclerosis, rheumatoid arthritis, psoriasis, juvenile
rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing
spondylitis, systemic lupus erythematosus, Hashimoto's disease, Graves
disease, inflammatory bowel disease, pancreatitis, Crohn's disease,
autoimmune diabetes, autoimmune ocular disease, ulcerative colitis,
irritable bowel syndrome (IBS), inflammatory bowel disease (IBD),
uveitis, or scleritis. [0252] 11. The method of any of paragraphs 1-10,
wherein the SGK1 inhibitor is a small molecule, a blocking antibody or
antigen-binding fragment thereof, a polypeptide, an antisense
oligonucleotide, an RNA molecule, an aptamer, or a ribozyme. [0253] 12.
The method of paragraph 11, wherein the wherein the small molecule is a
small molecule of Formula (I):

##STR00037##

[0253] wherein R1 is optionally substituted phenyl, optionally
substituted β-napthyl, or optionally substituted 3-CN-phenyl;
wherein R2 is CO2R4 or C(R4,R5) CO2R4; wherein R3 and R4 are
independently absent, H, C1-C6 alkyl, or C5-C8
cycloalkyl; each of which may be optionally substituted; wherein R5 and
R6 are independently absent, H, or C1-C6 alkyl, each of which
may be optionally substituted; and pharmaceutically acceptable salts
thereof. [0254] 13. The method of paragraph 11, wherein the small
molecule is a small molecule of Formula (Ia):

[0254] ##STR00038## [0255] 14. The method of paragraph 13, wherein R1
is phenyl, R2 is CO2H, and R3 is H. [0256] 15. The method of
paragraph 13, wherein R1 is phenyl, R2 is CO2H, and R3 is

[0256] ##STR00039## [0257] 16. The method of paragraph 13, wherein R1
is phenyl, R2 is CO2H, and R3 is

##STR00040##

[0258] 17. The method of paragraph 13, wherein R1 is phenyl, R2 is
CO2H, and R3 is

##STR00041## [0259] 18. The method of paragraph 13, wherein R1 is
β-napthyl, R2 is CH2CO2H, and R3 is H. [0260] 19. The
method of paragraph 13, wherein R1 is β-napthyl, R2 is

##STR00042##

[0260] and R3 is H.

[0261] 20. The method of paragraph 13, wherein R1 is β-napthyl,
R2 is

##STR00043##

[0261] and R3 is H.

[0262] 21. The method of paragraph 13, wherein R1 is phenyl, R2 is

##STR00044##

[0262] and R3 is H.

[0263] 22. The method of paragraph 13, wherein R1 is 3-CN-phenyl, R2
is

[0275] wherein R1 is optionally substituted phenyl, optionally
substituted β-napthyl, or optionally substituted 3-CN-phenyl;
wherein R2 is CO2R4 or C(R4,R5) CO2R4; wherein R3 and R4 are
independently absent, H, C1-C6 alkyl, or C5-C8
cycloalkyl; each of which may be optionally substituted; wherein R5 and
R6 are independently absent, H, or C1-C6 alkyl, each of which
may be optionally substituted; and pharmaceutically acceptable salts
thereof. [0276] 35. The use of the SGK1 inhibitor of paragraph 33,
wherein the small molecule is a small molecule of Formula (Ia):

[0276] ##STR00047## [0277] 36. The use of the SGK1 inhibitor of
paragraph 35, wherein R1 is phenyl, R2 is CO2H, and R3 is H. [0278]
37. The use of the SGK1 inhibitor of paragraph 35, wherein R1 is phenyl,
R2 is CO2, and R3 is

[0278] ##STR00048## [0279] 38. The use of the SGK1 inhibitor of
paragraph 35, wherein R1 is phenyl, R2 is CO2H, and R3 is

[0279] ##STR00049## [0280] 39. The use of the SGK1 inhibitor of
paragraph 35, wherein R1 is phenyl, R2 is CO2H, and R3 is

[0280] ##STR00050## [0281] 40. The use of the SGK1 inhibitor of
paragraph 35, wherein R1 is β-napthyl, R2 is CH2CO2H, and
R3 is H. [0282] 41. The use of the SGK1 inhibitor of paragraph 35,
wherein R1 is β-napthyl, R2 is

##STR00051##

[0282] and R3 is H.

[0283] 42. The use of the SGK1 inhibitor of paragraph 35, wherein R1
is β-napthyl, R2 is

##STR00052##

[0283] and R3 is H.

[0284] 43. The use of the SGK1 inhibitor of paragraph 35, wherein R1
is phenyl, R2 is

##STR00053##

[0284] and R3 is H.

[0285] 44. The use of the SGK1 inhibitor of paragraph 35, wherein R1
is 3-CN-phenyl, R2 is

[0287] This invention is further illustrated by the following examples
which should not be construed as limiting.

EXAMPLES

[0288] Upregulation of SGK1 during TH17 Cell Differentiation

[0289] The correlation between the SGK1 expression level during T cell
differentiation was previously unknown, so SGK1 mRNA level was first
measured under different conditions during T cell differentiation (FIG.
1). The data shown herein clearly demonstrate that TGF-β can
upregulate the expression level of SGK1. Moreover, addition of IL-6
together with TGF-β further increased the expression SGK1. IL-23, an
IL-12 family cytokine, is essential for enhancing generation of TH17
cells. It was further found that IL-23 further enhances the expression of
SGK1 (FIG. 1). These data indicated that SGK1 is specifically induced in
TH17 differentiation conditions, and can play a role in differentiation
of TH17 cells.

[0290] The time dependent expression of SGK1 during TH17 differentiation
was further examined by culturing naive CD4+ T cells under TH17
differentiating conditions (TGF-β plus IL-6) for 96 hours. After 48
hours, TH17 cells were supplemented with IL-23 until the end of the
culture period. It was found that expression of SGK1 was rapidly induced
after 2 hours and dropped down to baseline levels after 8 hours (FIG. 2).
However, IL-23 further induced SGK1 expression (FIG. 2). These results
indicate that TH17 differentiating condition (TGF-β plus IL-6) can
induce SGK1 expression, which is further sustained and stabilized by
IL-23.

Impaired TH17 Cell Differentiation in Absence of SGK1

[0291] Based on the demonstrations shown herein of regulation of SGK1
expression during TH17 differentiation, Sgk1.sup.-/- T cells were tested
for the ability to differentiate into TH17 cells. Naive CD4+ T cells from
SGK1 deficient and wild-type mice were differentiated into TH1, TH2 and
TH17 cells. It was found that upon stimulation with TGF-β and IL-6,
wild-type T cells differentiate into TH17 cell (˜22%). However,
SGK-deficient T cells showed significantly reduced IL-17 expression
(˜12%) (FIG. 3). However, TH1 and TH2 differentiation did not
change in the absence of SGK1, demonstrating the specificity of SGK1 for
TH17 differentiation (FIG. 3).

[0292] It has been shown that exposure of IL-23 is essential for expanding
and stabilizing TH17 cells. The data described herein, as shown in, for
example, FIG. 1, clearly demonstrates that IL-23 also induces the
expression of SGK1. It was next tested whether SGK1 is essential for
IL-23-dependent expansion of TH17 cells. Naive CD4+ T cells were sorted
from wild-type and SGK1-deficient mice and were cultured under TH17
differentiation conditions, as described herein. After a first round of
stimulation, cells were rested for two days in a cytokine-free medium.
Two days later, cells were activated in the presence or absence of IL-23,
and intracellular cytokine staining was performed for IL-17 expression.
IL-23 was able to expand already differentiated wild-type TH17 cells
(11.5 to 13%), however SGK1-deficient TH17 cells failed to expand in the
presence of IL-23 (˜4% to ˜1%). It was further tested whether
IL-23 can expand sorted memory TH17 cells. Both wild-type and
SGK1-deficient CD4+CD62L.sup.- cells were sorted and cultured with
either anti-CD3 alone or anti-CD3 plus IL-23. IL-23 clearly enhanced the
expression of IL-17A and IL-17F in wild-type cells, however
SGK1-deficient memory cells were defective in inducing expression of
IL-17A and IL-17F (FIG. 5). Altogether, the data shown herein clearly
demonstrates that SGK1 is essential for IL-23 dependent expansion of TH17
cells.

EAE Development in the Absence of SGK1

[0293] To study the function of SGK1 in TH-17-mediated autoimmune
responses, the susceptibility of wild-type and SGK1-knockout mice to EAE
induction can compared. Experimental allergic encephalomyelitis (EAE) is
an in vivo model for multiple sclerosis, an autoimmune disease of the
brain in humans, the pathology of which has been shown to be mediated by
TH17 cells. Mice can be immunized by subcutaneous injection of a peptide
consisting of amino acids 35-55 of myelin oligodendrocyte glycoprotein
(MOG(35-55)) in complete Freund's adjuvant (CFA) and pertussis toxin.
Wild-type mice develop a monophasic disease characterized by ascending
paralysis 9-16 d after immunization and prominent leukocyte infiltration
and microglial activation in the CNS. MOG-specific T cells that are able
to produce IL-17 and IFN-γ are found in the spleens of wild-type
mice, and MOG-specific T cells that produce IL-17 and IFN-γ are
found in the brains of wild-type diseased mice after 20 days. SGK1
knockout mice can be compared with wild-type mice in terms of the time of
onset (kinetics) of paralysis, if any, following immunization; the degree
or severity of paralysis, or other disease parameters, if any, following
immunization; and the number and degree of infiltration of TH17 cells in
the central nervous system (CNS). In mice having impairment of
TH17-mediated immune responses, parameters that measure the degree of
disease severity in EAE models are found to be inhibited or reduced. The
measurement of EAE severity under TH17-inducing conditions is reviewed in
"Th17 cells in autoimmune demyelinating disease." Segal B M. Semin
Immunopathol. 2010 March; 32(1):71-77).